technology poster single-cell pb 2020 dpi 041420 view · title: technology poster single-cell...
TRANSCRIPT
Key Yellow highlights indicate the target of the protocol
Single-Cell For all you seq...RNA Low-Level Detection
DP-SeqDesigned Primer-based RNA-se-quencing strategy (DP-seq)
DNAcDNA
AA(A)n
De�ne set of heptamer primers
Poly(A) selection First strand cDNA synthesis
Hybridize primers PCR
AA(A)n TT(T)n
No secondary structure
Unique sequenceAA(A)n
TT(T)n
Digital RNAHiRes-SeqFREQ-SeqRNAtag-Seq
cDNAcDNA1
cDNA2
cDNA1
cDNA2
Amplify SequenceUnique molecular barcodes are added after cDNA synthesis for quantitative allele frequency detection. High-resolution RNA-seq to assess noncoded base substitutions in mRNA (HiRes-Seq)
Adapters with unique barcodes
Align sequences and determine actual ratio based on barcodes
Some fragments amplify preferentially
True RNA abundance
cDNA1
cDNA2
mRNASmart-SeqNanoCAGE AAAAAAA
mRNA fragment
AAAAAAA
Second strand synthesis
AAAAAAAT T T T T T T
DNA
T T T T T T T
Adapter
AdapterSwitch mechanism at the 5’ end of RNA templates (Smart)
PCR ampli�cation PurifyFirst-strand synthesis with MMLV reverse transcriptase
CCCCCC
mRNAUMI Method AAAAAAA
mRNA fragment
AAAAAAA
First strand synthesis Second strand synthesis
AAAAAAAT T T T T T T
P7
True variant
Random errorDNA
T T T T T T T
P5
Index
Degenerate molecular tag (N10)
Unique molecular identi�ers (UMIs) uniquely identify copies derived from each molecule
PCR ampli�cation Align fragments from every unique molecular tag
CCC CCC
mRNASmart-Seq2 AAAAAAA
mRNA fragment
AAAAAA
cDNA synthesis Tagmentation
AAAAAA AAAAAAT T T T T T T T T T T TAdapter
Switch mechanism at the 5’ end of RNA templates (Smart)
PCRFirst-strand synthesis with MMLV reverse transcriptase
CCCCCC GGGTem-plate-switch-ing oligo
Locked nucleic acid (LNA)
CCCGGG
Enrichment-ready fragment
P5 P7
Index 1Index 2
Gap repair, enrich-ment PCR and PCR puri�cation
STRT
Single-cell tagged reverse transcription (STRT)
AA(A)n
AA(A)n
AA(A)n
AA(A)n
AA(A)n
AA(A)nCell 1
Cell 2
Cell 3
TT(T)n
TT(T)n
TT(T)n
AA(A)n
AA(A)n
AA(A)nTT(T)n
TT(T)n
TT(T)n
CCC
CCC
CCC
cDNA synthesis
Add 3 to 6 cytosines
TT(T)n
TT(T)n
CCC
CCC
CCCGGG
GGG
GGG
Template-switch-ing primer
Introduce unique index
Add oligo(dT) primer Pool Single-primer PCR and purify
Separate cell sequences based on unique indices
Cell 3
Cell 2
Cell 1TT(T)n
Unique index
5’ adapter
GGG
CEL-SeqAA(A)n
AA(A)n
AA(A)n
AA(A)n
AA(A)n
AA(A)n
AA(A)n
AA(A)n
AA(A)nCell 1
Cell 2
Cell 3T7promoter
Unique index
5’ adapter
TT(T)n
TT(T)n
TT(T)n
TT(T)n
AA(A)n
AA(A)n
AA(A)nTT(T)n
TT(T)n
TT(T)n
Second strand RNA synthesis
Fragment, add adapters and reverse-transcribe
Separate cell sequences based on unique indices
PoolCell 3
Cell 2
Cell 1
Cell expression by linear ampli�ca-tion and sequencing (CEL-Seq)
PCR
cDNA synthesis TagmentationPCRFirst strand synthesis
AAAAAAT T T T T T
AdapterCCC AAAAAA
T T T T T TCCCGGG
CCCGGG
Enrichment-ready fragment
P5 P7
Index 1Index 2
Gap repair and PCR
Single-nuclei RNA sequencing (snRNA-seq)
snRNA-Seq AA(A)n
Singlecell polyA RNA
Cell suspension
Lyse and centrifuge
Sort nuclei
Supernatant
NucleiNucleus
cDNA synthesis TagmentationPCRFirst-strand synthesis
AAAAAAT T T T T T
AdapterCCC
AAAAAAT T T T T TCCC
GGGCCCGGG
Enrichment-ready fragment
P5 P7
Index 1Index 2
Gap repair and PCR
Fixed and recovered intact single-cell RNA (FRISCR)
FRISCR AA(A)n
Fixedsingle cell polyA RNA
Cell suspension
Fix Sort single cells
Isolate RNA
Lyse cells and reverse crosslink
AAAAAA
Quartz-SeqWhole-transcript ampli�-cation for single cells(Quartz-Seq)
AA(A)n AAAAA AAAAAT T T T T
TTTTT T7 PCR
Add poly(A) primer with T7 promoter and PCR target
AAAAAT T T T T
Reverse transcriptionand primer digestion
T7 PCR T7 PCR
Poly A addition and oligo dT primer with PCR target
Generate second strand
Add blocking primer
Enrich with suppres-sion PCR
T T T T TPCR
T T T T T T7 PCRAAAAAT T T T TPCR AAAAA
TTTTT T7 PCRAAAAA
Blocking primer with LNA
cDNA
MARS-SeqMassively parallel RNA single-cell sequencing framework (MARS-Seq)
AA(A)n AAAAA
TTTTTT7UMI
Add poly(A) primer with partial T7 promoter and UMI
Second strand synthesis
RNA fragmentation RNA to ssDNA ligationDNaseI Reverse transcription
PCR and puri�cation cDNA
AAAAAT T T T T
T7UMI
partial rd1rev
P5 P7
scRNA-seqSingle-cell mRNA sequencing (scRNA-seq)
AA(A)n AAAAA
Add polyT primer
Reverse-transcribePoly(A)-tailed mRNA
Reverse transcription and primer digestion with ExoSAP-IT
PCR ampli�cation Shear DNA
AAAAAT T T T T
T T T T T AAAAAT T T T T
T T T T T AAAAAT T T T T
T T T T TAAAAA
Corrected sequenceIdentify low abundance RNA viruses with circular sequencing (CirSeq)
CirSeqWhole-genome RNA
AA(A)n
Circularize RNA with kinase and RNA ligase 1
Random primers
Circular RNA template
Repeat 1Repeat 2
Repeat 3
Repeat 1Repeat 2Repeat 3
Mutation
Error
Transcriptome in vivo analysis (TIVA)
TIVA Whole-genome RNAAA(A)n
Capture on Streptavidin coated magnetic beads
mRNA from single cell
AAAAAAA AAAAAAA
UUUUUUUUUUUUUUUUUUCy3
Cy5 PLCPP
Biotin
PLS S
CPP Cell-penetrating peptide
Disul�de bondS SPhotocleavable linkerPL
UUUUUUUUUUUUUUUUUUCy3 UUUUUUUUUUUUUUUUUUCy3
CPP SAAAAAAA AAAAAAA
UUUUUUUUUUUUUUUUUUCy3
Cy5 PLPL
S
UUUUUUUUUUUUUUUUUUCy3
AAAAAAA AAAAAAA
Cy5 PL PLS AAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAA
Load into cells
CPP peptide released
Photoactivate Anneal to mRNA
Cell
Gene expression cytometry (CytoSeq)
CytoSeqBarcoded mRNA from single cells
AA(A)n
Singlecell
Cell suspension
Each bead with unique oligos
Load cells and beads into microwells
Cell lysis, mRNAshybridize on bead
Pool all beads from microwells
cDNA synthesis and ampli�cation
Sequence
UniversalCell labelMolecular indexOligo(dT)
Analyze mRNA transcripts from individual cells in droplets (Drop-seq)
Drop-Seq
Barcoded mRNA from single cells
AA(A)n
Singlecell
Cell suspension
Each bead with unique oligos
Load cells and beads into droplets
Cell lysis, mRNAshybridize on bead
Pool all beads from droplets
cDNA synthesis and ampli�cation
Sequence
UniversalCell labelMolecular indexOligo(dT)
TCRα mRNA
TCRβ mRNA
Oil emulsion
Identify T-cell Receptor (TCR) alpha–beta chain pairing in single cells
Reverse transcription
Ampli�cation Overlap extension Blocker primers
Nested PCR ampli�cation
TCR Chain Paring
AA(A)n
AA(A)n
TCRα
TCRβ
TCRα
TCRβ
TCRα TCRβ TCRα TCRβTCRα TCRβ
DNAPCR suppression of non-fused molecules
CDR3α CDR3β
CDR3
Whole-genome RNA
Peptide nucleic acid-assisted identi�cation of RNA binding protein (PAIR)
PAIR
Capture on magnetic beads
Visualize protein on SDS-PAGE
CPP Cell-penetrating peptide
Disul�de bondS SPhoto-activatible compoundBpa
Binding site for RNA-bind-ing protein
Create peptide nucleic acid analogs (PNAs)
AA(A)n AA(A)n
PNACPP S S
PNABpa
CPP peptide released
BpaCPP S
PNABpaS
AA(A)n AA(A)nS S Bpa
PhotoactivateLoad into cells
AA(A)n
Cell labeling via photobleach-ing (CLaP)
CLaPBarcoded mRNA from single cells
AA(A)n
Singlecell
Con�uent cells in culture
Biotin-4-�uo-rescein (B4F)
Photobleach and crosslink with 473 nm laser
Cy5-streptavidin labeling
Tagged cells isolated, reverse-transcribed and sequenced
Rinse
TCR-LA-MC PCR TCR ligation-anchored-mag-
netically captured PCR (TCR-LA-MC PCR)
Constant (C) gene of the TCR chains
(C) gene cDNA synthesis
RNA digestion
ssDNA linker ligation
PCR 1
C
RNAC
P5 P7
Index 1Index 2
PCR 2 with nested primers
Add sequencing adapters
DNA ready for sequencing
CCCCCCCCCCCCCCCCCCCC
MALBAC primers
RNAUMI
cDNA
PolyA RNASingle cell
Non-polyA RNA
Reverse transcription
RNA digestion and dC tailing
G-enriched MALBAC primers with UMIs
GGGGGGGGGGCCCCC
Cell lysis
Repeat 9x
Multiple annealing and dC-tailing-based quantita-tive scRNA-Seq (MATQ-Seq)
MALBAC primers initiate quasilinear ampli�cation to reduce ampli�cation of PCR bias
Primer hybridization at low temperatures increases RT e�ciency and non-polyA transcript detection
Addition of dC tail to 1st cDNA strands increases e�ciency of 2nd strand synthesis
UMIs drastically reduce exponential PCR ampli�cation bias
Library prep and sequence
AA(A)nMATQ-seq
Random displacement ampli�cation sequencing (RamDA-Seq)
Strand displacement ampli�cation assisted by gp32
2nd cDNA strand synthesis with NSR primers and Klenow Fragment
Tn5 transposase-mediated library prep
Random nicks on cDNA by DNase I
Not-so-random (NSR) primers prevents binding with rRNA
1st cDNA strand synthesis with RNase H minus RTase
AA(A)n RNA
cDNA3’5’
DNase Igp32
Non-polyA RNA
PolyA RNANSR primerCell
lysisSingle cell
NSR primerRamDA-seq
AA(A)n
AA(A)n
Singlecell
Singlecell
Nuclei
Microchannel containing nuclei
Microchannel containing cytoplasm
Cytoplasm
Lysis and ITP extraction
Load into micro�uidics system
ITP acceleration isolates cytRNA
nucRNA-Seq by SMART-Seq2
Custom micro�uidics with isotachophoresis (ITP) bu�er chemistry
cytRNA-Seq by SMART-Seq2
Nuclear RNA
Cytoplasmic RNASequencing-ready
cDNA
Sequencing-ready cDNA
SIngle-cell integrated nucRNA & cytRNA sequencing (SINC-Seq)
Correlation of gene expression between nuclei and cytoplasm from a single-cell
Nuclei is prevented from mixing with cytoplasmic material by hydrodynamic traps. Voltage manipulation physically separates cytoplasmic material following cell lysis
SINC-seq
Virus-infected single cell
Host RNA
Viral RNA
Virus-inclusive single-cell RNA-Sequencing (viscRNA-Seq)
Virus-infected cells are sorted by FACS into 384 well-plates and lysed
Host mRNA are captured using polyT oligos. Viral DNA captured by virus-speci�c oligos
5’-blocked template-switching oligos are used to reduce formation of concatemeric artifacts
AA(A)n Host RNA cDNA
Sequencing -ready cDNA
Well containing single-cell
Viral RNAAA(A)n
Infected cellsFACS sort into well plates Lyse
RT and template-switching Amplify and
library prepCapture RNA
Modi�ed SMART-Seq2
viscRNA-seq
AAAAAT T T T T AAAAA T T T T T AAAAA T T T T T
T T T T TPolyA RNA
AA(A)n
Single-cell in PCR well plate
Cell barcode
UMI
cDNA
mRNAPCR primer
PolyA tail synthesized by TdT enzyme
PCR primer with polyT tail
2nd strand synthesis
cDNA library ready for sequencing
Single cell Lysis & RT
Pool & purify
Amplify & custom library prep
High-throughput single-cell RNA-Seq method that e�ectively uses limited sequence reads (Quartz-Seq2)
Single cells are sorted into PCR well plates containing lysis bu�er and RT primers (cell barcode, UMI, and oligo dT)
WTA using terminal deoxynucleotidyl transfer-ase (TdT) provides 3.6 fold increase in polyA tagging to UMI conversion e�ciency
Custom library prep attaches sequencing adapters and pool barcodes to enable mixing of di�erent sets of cell barcode-labeled cDNA
Quartz-seq2
Fixed cells
RNA
cDNA
Barcode 1 Barcode 2 Barcode 3 Barcode 4
Pool
RT with 1st barcode set
Ligate 2nd barcode set
Ligate 3rd barcode set
Amplify with 4th barcode set
PCR of cDNA using 4th well-speci�c barcode along with sequenc-ing adaptors
Fixed and permeabilized cells are reverse-tran-scribed using microwell- speci�c unique barcodes (yellow for this well)
Each cell can be distin-guished by unique barcode sequences attached to cDNA
Barcode 3 contains UMIs and conjugat-ed biotin molecule
Ready for library prep
Random split
Random split Lysis and
split
Random split Pool PoolBiotin
Single cell
Split-pool ligation-based transcriptome sequencing (SPLiT-Seq)
PolyA RNAAA(A)n
SPLiT-seq
UMI
2nd index tagging and sequencing adapters
Well-speci�c transposomes
i5 adapter with cell index
i7 adapter with cell index
PoolIndex 1
cDNA
Index 2
UMISingle cell
Single-cell combinatorial indexed RNA sequencing (sci-RNA-Seq)
Fixed cells or nuclei are randomly sorted and reverse transcribed using polyT adapters with UMIs and well-speci�c index
cDNA strands are tagged with another set of well-speci�c barcodes that includes i7 and i5 sequencing adapters
Ready for sequencing
RT with 1st barcode set
Fixed cellsRandom split
Random splitPool 2nd strand
synthesisPolyA RNAAA(A)n
TTTT
TTTT
TTTT
sci-RNA-Seq
Each bead has unique cell-identifying oligo sequence
Load nuclei and beads into droplets
Break droplets & pool beads
cDNA synthesis and ampli�cation
OligobeadsDroplets
Droplets (enlarged)
Sequencing-ready cDNA
PCR handleCell barcodeUMIOligo(dT)
AAAAAAAA
AAAA
mRNA
Single nuclei
Load nuclei into droplet micro�uidic system
Single nuclei droplet-based sequencing (snDrop-Seq)
PolyA RNA
Nuclei suspension
Single nuclei
AA(A)n
Nuclei lysissnDrop-Seq
Massively parallel single-nucleus RNA sequencing with droplet technology (DroNc-seq)
AA(A)n
Cell suspension
Each bead has unique cell-identifying oligo sequence
Load cells and beads into droplets
Barcoded mRNA from single nuclei
Pool all beads from droplets
cDNA synthesis and ampli�cation
Sequence
LysePolyA RNA
Oligobeads
PCR handleCell barcodeUMIOligo(dT)
Single nuclei Isolate
nucleiAAAA
AAAA
AAAA
mRNA
non-polyA RNA
DroNC-Seq
DNA Low-Level Detection
MALBACGenome
Hybridize primers PCR
27-bp common sequence8 random nucleotides
Partial amplicons
Template
Denature
Denature
Hybridize primers Synthesis
Multiple annealing and looping-based ampli�cation cycles (MALBAC)
DNA
Cycles of quasilinear ampli�cation
Looped full amplicons
Bst DNA polymerase
Genomic DNA
GenesmMIP
Copy target sequence Exonuclease Corrected sequence
Align fragments from every unique molecular tag
Sample indexRead1
Read2
True variant
Random errorSingle Molecule Molecular Inversion Probes (smMIPs) for detecting low frequency targets
PCR ampli�cation
Degenerate molecular tag
Targeted STR
Short tandem repeat (STR)MIPSTRCopy target STR Amplify and sequenceTargeted capture of STR
loci by smMIPs (MIPSTR)
Degenerate molecular tag Strain I
Strain IIStrain I
Strain INatural variation between individuals Somatic variation
within an individual
Nuc-SeqSNES
Cell 1
Cell 2
Cell 3
Cell sorting from G2/M distribution
Lyse cell NucleusSingle G2/M nucleus sequencing of cells in S phase (nuc-seq)Single nucleus exome sequencing (SNES)
Single cell genome
phi29 Limited ampli�cation S1 nucleaseSynthesis DNA
GenomeMDAIMS-MDAMIDAS
Primer hybridization
Nascent replication fork
phi29 phi29 S1 nuclease Ampli�ed DNA
3’ blocked random hexamer primers
Synthesis SynthesisMultiple displacement ampli�cation (MDA) Immunomagnetic separation for targeted bacterial enrichment for MDA (IMS-MDA) Microwell displacement ampli�cation system (MIDAS)
Cell lysis
Size select
Break emulsion, RT, & template switching
Each antibody tagged with unique barcode sequence to allow sequence-based antibody ID
Antibodies bind to cell-surface proteins on cells
Single-cells and beads are loaded into droplets
mRNA and oligos from antibodies both have polyA tails, here they anneal to oligobead at its polyT end
dscDNA from cellular mRNA are considerably longer than dscDNA from antibody-oligo complex sequences
Cell-speci�c transcripts are identi�ed by barcode sequences and couple gene expression data with cell-surface protein marker data
Cellular indexing of transcriptomes & epitopes by sequenc-ing (CITE-Seq)
AntibodyAntibody barcode
Single cell
CellsLoad into droplets
PolyA RNA
Surface protein
AA(A)n
AAAAAAAA
AAAA
AA(A)nS S
OligobeadsAntibody-oligo complex
PCR handleCell barcodeUMIOligo(dT)
mRNA
dscDNA
From cellular mRNA
From antibodies
non-polyA RNA
CITE-Seq
Single-cells and beads are loaded into droplets
mRNA and oligos from antibodies both have polyA tails, here they anneal to oligobead at its polyT end
dscDNA from cellular mRNA are considerably longer than dscDNA from antibody-oligo complex sequences. sgRNAs have their own PCR primers
Transcripts can be identi�ed to cellular level through its cell-barcodes, connecting multiple modalities within a single experiment
Single cell PolyA RNA
Protein markers & sample tagging
AA(A)n
Sample A
Sample B
Sample C
Oligobeads
AntibodyAntibody barcode
AA(A)nS S
Antibody-derived tags
Antibody
AA(A)nSample A barcode
Antibody
AA(A)nSample B barcode
Antibody
AA(A)nSample C barcode
Cell lysis
AAAAAAAA
AAAA
mRNA
non-polyA RNA
Load into
drop-lets
Size select
From cellular mRNA
From antibodies
From sgRNAs
Break emulsion, RT, & template switching
dscDNA
Sample tagging to ubiquitous protein markers
Epitope-speci�c tagging Droplet microfuildics
Pool
Expanded CRISPR-compatible cellular indexing of transcriptomes and epitopes by sequencing (ECCITE-Seq)
Hashtag oligos (HTOs) target speci�c proteins with barcodes to provide sequence-based cellular identi�cation of sample origin.
Antibody-derived tags mark cellular antigens of interest
gRNA
ECCITE-Seq
PCR handleCell barcodeUMIOligo(dT)
AA(A)n
AAAAAAAA
AAAA
PolyA RNAgRNA
CRISPR/Cas9Single cell
Cell suspension gRNA
construct
Drop-Seq
Lentiviral transduction & enrichment
AAAA
AA
AA
AAAA
AAAA
mRNA
gRNA
gRNA
Transcriptome
Each bead has unique cell-identifying oligo sequence
Puromycin selection enriches successful gRNA-transduced cells
Load cells and beads into droplets
scRNA-seq directly link transcriptome pro�le with corresponding guide RNA
Lyse
cDNA libraryBreak droplets & library prep
Pooled CRISPR screens with single-cell transcrip-tome resolution (CROP-Seq)
Custom lentiviral construct contains gRNA in polyA mRNA transcripts so they can be detected through scRNA-Seq
CROP-Seq
Oligobeads
PCR handleCell barcodeUMIOligo(dT)
AA(A)n
AAAAAAAAAAAAPolyA RNAgRNA
CRISPR/Cas9Single cell
Cell suspension
gRNA constructViral
infection & enrichment
gRNA
Transcriptome
Puromycin selection enriches successful gRNA induced cells
scRNA-seq directly link transcriptome pro�le with corresponding guide RNA
cDNA libraryBreak droplets & library prep
Mosaic single-cell analysis by indexed CRISPR sequencing (Mosaic-Seq)
Cellular enhancer activity perturbed by viral transduction of gRNAs, followed by antibiotic selection
Mosaic-Seq
Drop-Seq
AAAA
AA
AA
AAAA
AAAA
mRNA
gRNA
Each bead has unique cell-identifying oligo sequence
Load cells and beads into droplets
Lyse
Oligobeads
PCR handleCell barcodeUMIOligo(dT)
Minimize arti�cially induced transcrip-tional perturbations followed by scRNA-Seq (Act-Seq)
Actinomycin D (ActD) inhibits dissocia-tion-induced gene expression common in conventional dissociation methods
Each bead has unique cell-identifying oligo sequence
Load nuclei and beads into droplets
Break droplets & pool beads
cDNA synthesis and ampli�cation
OligobeadsDroplets
Droplets (enlarged)
Sequencing-ready cDNA
PCR handleCell barcodeUMIOligo(dT)
AAAAAAAA
AAAA
mRNA
Load into droplet micro�uidic system
Single cell
Cell lysispolyA RNA
Singlecell
Frozen tissue slice Drop-SeqCell dissociation followed by Actino-mycin D treatment
AA(A)n
Act-Seq
PDMS subnanoliter array Oligobeads
Cell suspension
mRNA
Simple, portable platform for massively parallel scRNA-Seq (Seq-Well)
Individual cells trapped in PDMS nanoarray wells
All oligos in the same bead contain identical cellular barcodes but unique UMIs
PolyA RNA captured by oligobeads
Wells are sealed with a semi-permeable membrane
Barcoded cDNA library
Cell lysis
Captured mRNA prepared for sequencing
polyA RNA
Singlecell
AA(A)n
UniversalCell labelMolecular indexOligo(dT)
Seq-Well
Agarose microwell array Oligobeads
Cell suspension
Single-cell
mRNA
mRNAAdaptors
Transcriptomic pro�les for thousands of single-cells (Microwell-Seq)
Individual cells trapped in agarose microarray wells
All oligos in the same bead contain identical cellular barcodes but unique UMIs
PolyA RNA captured by oligobeads
Barcoded cDNA library
Cell lysis
Captured mRNA enriched using SMART-Seq2
polyA RNA
Singlecell
AA(A)n
UMICell labelMolecular indexOligo(dT)
Microwell-seq
Nanogrid single-nuclei RNA sequencing (Nanogrid SNRS)
Nanowells on the nanogrid are lined with custom oligos for polyA RNA capture and cDNA synthesis
Stained nuclei or cells are loaded into nanowells by nanodispensor
Viable vs. nonviable wells sorted by using �uorescence imaging
Fluorescent microscopy imaging
polyA RNASingle cell or nuclei
AA(A)n
Nanogrid
Oligo-lined nanowells
None/dead in well
More than 1 per well
1 nucleus/cell per wellDAPI-stained
nuclei
DispenseSelect viable wells & lyse
RT, template switching, and one-sided P7 tagmentation
Barcoded cDNA library �anked by sequencing adapters
cDNA library generation
P5 adapter
Well barcode
UMI
Oligo(dT)
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mRNA
cDNANanogridSNRS
PoolscRNA-Seq work�ow
Singlecell
Cell cultures with di�erent treatments
LMO-labeled cellsLipid-modi�ed oligonucleotide (LMO) with barcode
LMO sample barcodes are identical within a treatment group but unique among di�erent treatments
Rapid, modular, and universal scRNA-Seq sample multiplexing strategy using lipid-targeted indices (MULTI-Seq)
LMOs remain adhered to surface membranes until cell lysis during scRNA-Seq
Sequence analysis of LMO sample barcodes can identify treatment group, while cellular barcodes can identify individual cells
Sample barcode
5’ PCR handle
mRNAAA(A)n AAAA AAAAMULTI-Seq
Hybridize primers
Nascent replication fork
phi29 phi29 S1 nuclease Sequence and feed into SCcaller
3’ blocked random hexamer primers
Synthesis Synthesis
Genome
Perform in low temperatures
Single cell
Cell suspension Isolate
single-cells & lyse
Single-cell multiple displacement ampli�cation (SCMDA) with single-cell variant caller (SCcaller)
SCMDA
Linear ampli�cation via transposon insertion (LIANTI)
Single-cells lysed and gDNA tagmented by the LIANTI transposome
T7 RNA polymerase binds to T7 promoter site on tagmented gDNA
Linear ampli�cation of gDNA signi�cantly reduces ampli�ca-tion bias and replication errors
RT
RNase digestion and 2nd strand cDNA synthesis and UMI barcoding
Genome
gDNA
gDNA RNA cDNAUMIcDNA/RNA hybrid
LIANTI Transposome
LyseTransposase
T7 RNA Polymerase
Transposase bind site
T7 Promoter
Single cellLIANTI
Pool Pool
Transposase-based tagging
Random sorting
Isolate nuclei and nucleosome depletion
TransposomeDNA
DNASingle cell
PCR-based tagging
Random sorting
Single-cell combinatorial indexed DNA sequencing (sci-DNA-Seq)
Nucleosome depletion via SDS cross-linking
Randomly sorted nuclei are tagmented with well-speci�c unique indexed adapters
15-25 cells are randomly sorted into wells for 2nd round set of PCR-based indexing
2 index combination uniquely identi�es DNA strands from the same single-cell
sci-DNA-Seq
Single cell RNA barcoding and sequencing (SCRB-Seq)
SCRB-Seq AA(A)n
Single cell
Cell suspension
Cell sorting by FACS
Cell lysis Isolate RNA
AA(A)nAA(A)nT T (T)n
AA(A)nTT(T)n
Add adapters and reverse-transcribe
cDNAPool PCR
Cell labelUniversal primer
Oligo(dT)
Second strand RNA synthesis
Hybridize oligo
High-throughput single-cell labeling (Hi-SCL)
Hi-SCLBarcoded mRNA from single cells
AA(A)n
Singlecell
Cell suspension
Each droplet with unique oligos
Insert oligos in droplets
Load single cells into droplets with lysis bu�er
Fuse droplets Pool all droplets cDNA synthesis and ampli�cation
Sequence
Universal primer
Oligo(dT) RT bu�er
High-throughput single-cell labeling with indexing droplets (inDrop)
inDropBarcoded mRNA from single cells
AA(A)n
Singlecell
Cell suspension
Each microsphere with unique oligos
Oligos attached to hydrogel
Load single cells into droplets with lysis bu�er
Combine micro-spheres and droplets
Pool all droplets
UV primer release
cDNA synthesis and ampli�cation
Sequence
Photocleavable linker
Oligo(dT)RT bu�er
Cell label
A single nucleus RNA-Seq method (Nuc-Seq)
Nuc-Seq AA(A)n
Singlecell
Tissue Fixation and freeze
Lyse and centrifuge
Sort nuclei
Nuclei mRNA fragment
AAAAAA
cDNA synthesis TagmentationPCR
AAAAAAT T T T T TCCC
GGGLocked nucleic acid (LNA)
CCCGGG
Enrichment-ready fragment
P5 P7
Index 1Index 2
Gap repair and PCR
Single-cell RNA barcoding and sequencing (SCRB-Seq)
Div-Seq AA(A)n
Singlecell
Tissue in vivo labeled with 5-ethynyl-2’-de-oxyuridine (EdU)
Nuclei isolation
Click-IT tagging
FACS sort
mRNA fragment
AAAAAA
cDNA synthesis TagmentationPCR
AAAAAAT T T T T TCCC
GGGLocked nucleic acid (LNA)
CCCGGG
Enrichment-ready fragment
P5 P7
Index 1Index 2
Gap repair and PCR
CH3O CH3
Display methods on mobile device
N
NN
N
O O
OH
NH
HN
H
H S
Biotin
Preparation of acylated RNA for biotin–streptavidin puri�cation. DIBO, dibenzocyclooctyne
N
O
N3
ORNA
N
O
ORNA
NN
N
Biotin
N
NN
O
N3
RNA +
Acylation
DIBO-biotin “click”
5-Ethynyl-2'-deoxyuridine (EdU)
NH
N
HO
HO
O
O
O
5-iodouridine (5IU)
NOH
O
OH OH
NH
O
IO
4-thiouridine (4SU)
NHO
O
OH OH
NH
O
S
5-bromo uridine (5BrU)
NOH
O
OH OH
NH
O
BrO
6-Thioguanosine (6SG)
NOH
O
OH OH
N NH2
NHN
S
Photoactivatable Nucleosides
Locked nucleic acid (LNA)
NOH
O
OH O
N NH 2
NHN
O
N
NH2
NO
N
NH2
NO
CH3
Cytosine 5-Methyl Cytosine
N
NH2
NO
CH3
5-Methyl Cytosine
N
NO
O
Uracil
Bisul�te conversion
N6-Methyladenosine (m6A)
NO O
O
O O
OH OH
N
NN
N
CH3H
P
O O
OH
NH
HN
H
H S
Biotin
Biotin-4-�uorescein (B4F)
O
O
NH
HN
H
H S
O
O
O
HO
HOHN
NHO
p-benzoylphenylalanine (Bpa)O
HO
O
H2N
SUPeR-seq
Single-cell universal poly(A)-indepen-dent RNA sequencing (SUPeR-seq)
AA(A)nAAAAA
Add poly(A) primer with T7 promoter and PCR target
Reverse transcription and primer digestion with ExoSAP-IT
PCR ampli�cation Puri�cation DNA
AAAAANNNNNT T T T T
NNNNNT15NNNNNT15
NNNNNT15
AAAAAT T T T T
AAAAAT T T T T
PolyA RNA
RNA
cDNA
RT primer with well barcode and UMI
Transposase contains well-speci�c barcodes
AA(A)n
AAAARNA
AAAATTTT
TTTT
Pool & sequence
Single-cell combinatorial indexing-based co-assay that jointly pro�les chromatin accessibility & mRNA (sci-CAR)
Nuclei are indexed for ATAC-Seq and RNA-Seq using well-speci�c barcodes
Addition of the 2nd well barcodes tags each nuclei with two di�erent combination of well barcodes, acting as a marker to contrast DNA fragments that are from di�erent cells
Open DNA
Single nuclei
Pool nuclei
Stain and FACS sort
2nd strand cDNA strand synthesis & nuclear lysis
Split wells into ATAC-Seq and RNA-Seq groups
Amplify DNA with RNA-Seq index
5000 nuclei/well
Split
Add RNA-Seq index
Add ATAC-Seq index
Nuclei suspension
25 nuclei/well
gDNA
in-situ tagmentation
DNA with ATAC-Seq index
DNA with RNA-Seq index
Seq adapters2nd well barcode
RNA-Seq dedicated lysate
Amplify DNA with ATAC-Seq index
DNA with ATAC-Seq index
DNA with RNA-Seq index
Seq adapters
2nd well barcode
ATAC-Seq dedicated lysate
sci-CAR
Integrated Techniques
Duplex-Seq α βVery rare mutation
Duplex sequencing detects rare mutations by sequencing and aligning both strands of the DNA
P5
P7 P5
P7
A mutation occurs on both strands
12 random base index
12 random base index
True variantRandom error
Ligate and PCR Rare variantSequence Create single strand consensus sequence from every unique molecular tag
ConsensusCreate duplex sequences based on molecular tags and sequencing primers
Add adapters
OS-Seq Gene
Target sequence
Adapter sequence
Flow cell
Sequencing Primers
Target sequence Single adapter library
Hybridize Hybridize
SequenceOligonucleotide-selective sequencing (OS-Seq) captures and sequence gene targets on the �ow cell
Create target-speci�c oligos Extend and Denature
Extend and Denature
Extend and Denature
Sequence reads 1 and 2
Fragment and add single adapters
Genome DNA and mRNA sequencing (DR-Seq)
DR-SeqAA(A)n
Singlecell polyA RNA
DNA
AA(A)n
polyA RNA
DNA
Single cell
RT with barcoded primerLyse cell Ad-2 primer
Split samples
Quasilinear ampli�cation
SequencegDNA ampli�cation
cDNA ampli�cationT T T T T T T T T TAAAAAAA
PCR and Remove adapters
2nd strand synthesis
Methylome and transcrip-tome sequencing from a single cell (scM&T-seq)
scM&T-Seq
Align RNA and methylome
AA(A)n
Singlecell polyA RNA
DNA
AA(A)n
polyA RNA
DNA
Cell suspension
Isolate single cell
Separate the DNA and the RNALyse cell Sequence
T T T T T T T T T TAAAAAAA
Streptavidin magnetic bead with mRNA capture primerStreptavidin magnetic bead with mRNA capture primer
T T T T T T T T T TAAAAAAA On-bead transcriptome
ampli�cation with Smart-Seq2Whole-genome ampli�cation with scBS-seq
Very rare mutationSafe-SeqS
DNA Shear
Mutation
Amplify and solid phase capture
SequenceSafe-sequencing system is a unique molecular identi�er (UMI) approach to detect rare variants (Safe-SeqS)
Adapter ligation Randomly sheared ends serve as UMIs
Align sequences and determine actual ratio
True mutant
scChIP-Seq
Exonuclease digestion Immunoprecipitation DNADNA-protein complex DNA extraction
Crosslink proteins and DNA Sample fragmentationSingle cell chromatin immunoprecipitation (scChIP-seq)
Single-cell triple omics sequencing (scTrio-seq)
scTrio-SeqAA(A)n
Singlecell polyA RNA
DNA
DNA methylation
Cell suspension
Isolate single cell
Lyse and centrifuge
Supernatant
Nucleus
AA(A)npolyA RNA
Add carrier RNA
AA(A)nT T (T)ncDNA synthesis PCR and sequenceAdd poly A with TDTHybridize oligo
AA(A)n
DNAAdd sequencing adapters PCR and sequence
Align sequencesMethylated regions
Methylated adapter
End repair and ligation
Bisul�te conversion
Converted fragments
MspI digestion
PCR and sequence
Methylated DNA
scAba-SeqDNADetect 5hmC marks in single cells
with AbaSI nuclease (scAba-seq)Glucosylated 5-hmC
5hmc residues
T4-βGTHydroxy-methyl-ated DNA
AbaSI Ligate Pool T7 ampli�cationPrimer
Illumina 5’ adapterT7 promoter
Adapter with cell-speci�c barcodeSingle cell
Droplet-based single-cell ChIP-seq (Drop-ChIP)
Drop-ChIPSingle cell
Barcoded sequences from single cells
Cell suspension
Droplet with unique oligos
Load single cells into droplets with lysis bu�er and MNase
Fuse droplets Pool all droplets SequenceChromatin immuno-precipitation
Single cell
scATAC-Seq(Microfluidics)
Fragmented and primed DNASingle-cell assay for transposase accessible chromatin (scATAC-Seq)
Lyse and introduce Tn5 transposase
Pool libraries from all cells
Amplify with cell-speci�c barcodes
Insert in regions of open chromatinCell suspension
Micro�uidics device
Isolate single cell
scRC-Seq
Genomic DNA Enriched library
Novel retrotrans-position events
Retrotransposon binding site
Single cell retrotransposon capture sequencing (scRC-Seq)
Cell suspension
FACS isolation
Pick nuclei
Whole-genome ampli�cation
Create sequencing library
Sequence captureNucleus
Single cellscATAC-Seq(Cell index)
DNASingle-cell assay for transposase accessible chromatin (scATAC-Seq)
Barcode each well with Tn5 transposase
Cell suspension Isolate Nuclei Split sample
Pool and dilute
Split sample PCR-barcode every well
Pool for library prep
SMDBSingle-molecule droplet barcoding (SMDB)
DNA templates Single template encapsulation
Template ampli�cation Template fragmentation Barcode every droplet Pool for library prep
DNA
gRNA e�ciency
i5 i7UMIgRNA
UMI-based pooled CRIPSR screening for single-cell lineage tracing and quanti�cation of gRNA e�ciency (CRISPR-UMI)
Each gRNA construct contains random 10 nt UMI barcode, gene-speci�c spacer sequence, and Illumina indexed adaptors. They are also �anked by PacI restriction sites
Strong limiting dilution isolates di�erent editing outcomes to its own colonies
Each cell in a colony was edited by speci�c gRNA hence contains the same UMI sequence
gRNA sequence is read and resulting data used to determine which gRNAs were most e�cient in creating the desired outcome
gRNA
CRISPR/Cas9Cell suspension
Cell
CRISPR-UMI gRNA design
Edit, select, expand Expand Purify DNA
and amplify
gRNA constructsLimiting
dilution
CRISPR-UMI
Single-cell dissection and UV catapulting into collection tubes
Topographic single-cell sequencing (TSCS)
Tissue imaging to capture spatial informa-tion
Single-cell whole-genome ampli�cation by DOP-PCR
Barcoded sequencing libraries
Each single-cell tagged by unique barcodes
Spatial information can be traced back using the barcodes
Single-cells in collection tubes
DOP-PCRCell-speci�c barcodes
DNA with spatial info
Frozen tissue slices
UV-catapulting Cell-speci�c barcodes
TSCSSingle cell
Random primer
Methylated DNA
Bisul�te conversion
Amplify and sequence
Single-nucleus methyl-cytosine sequencing (snmC-Seq)
Isolated single cell
Lyse
Random priming Extend Pool samples and Adaptase reaction
AdaptersnmC-Seq
Methylated DNASingle cell
Isolate nuclei and nucleosome depletion
Pool Bisul�te conversion, linear ampli�cation, and PCR ampli�cation
Transposase-based tagging
Random sorting
Transposome
PCR-based tagging
Random sorting
Single-cell combinatorial indexed for methylation analysis (sci-MET)
Tagmented with cytosine-deplet-ed adapters. Adapters contain well-speci�c index and read 1 primer sequence
This step converts unmethylated cytosines, tags fragments with a second well-speci�c barcode, and attaches sequencing adapters.
Sequencing-ready cDNA
1st adapterSequencing adapters + well-speci�c index
Pool
sci-MET
Cell barcodeT7 promoter
Tn5 bind sitePool
Pool &sequence
Single nuclei
Single-cell transposome hyper-sensitive site sequencing (scTHS-Seq)
SplitSplit
In-vitro transcription ampli�cation
3’ end transposition & end �ll-in
Sequencing adaptor ligation & amplify
Nuclei suspensionOpen DNA
2000 nuclei/well
gDNA
100 nuclei/well
3’ adaptors Illumina indexed adaptors
gDNA
Tn5059 transposome
scTHS-seq
Single cell
Release nuclei and Dpn II digest
Ligation of 1st barcode
Ligation of 2nd barcode
Ligation of barcoded bitotinylated double-stranded bridge adaptors
Proximity ligation
Ligation of custom-barcoded Y-adaptors with sequencing adaptors
PCR amplify and sequence
SplitFixed cells Well-plate
Protein-DNA complex
1st barcode
2nd barcode
Pool Pool & purifySplit
Single-cell combinatorial indexed Hi-C (sciHi-C)
Biotin
At most 25 cells per well
sciHi-C
Genome and transcriptome sequencing from a single cell (G&T-seq)
G&T-Seq
Align RNA and genome
AA(A)n
Singlecell polyA RNA
DNA
AA(A)n
polyA RNA
DNA
Cell suspension
Isolate single cell
Separate the DNA and the RNALyse cell Sequence
T T T T T T T T T TAAAAAAA
Streptavidin magnetic bead with mRNA capture primer
T T T T T T T T T TAAAAAAA On-bead transcriptome
ampli�cation with Smart-Seq2Whole-genome ampli�cation with MDA
AA(A)n
polyA RNA
Copy Number Alterations
DNA methylation
scBS-Seq
Supernatant
Nucleus
AA(A)npolyA RNA
Add carrier RNA
AA(A)nT T (T)ncDNA synthesis PCR and sequenceAdd poly A with TDTHybridize oligo
AA(A)n
DNAAdd sequencing adaptors PCR and sequence
Cellsuspension
Lyse and separate RNA from nucleus using magnetic bead and centrifugation
Single-cell triple omics sequencing version 2 (scTrio-Seq2)
Random primer 1
Methylated DNA Bisul�te conversion
Random primer 2
Align fragments from every UMI and sequence
First random priming
Second random priming
Repeat 4 times
PCRExtend
Adapter AdapterExo I and purify
Single cellscTrio-Seq2
CpG dinucleotides Methylated CpG
Single-cell nucleosome, methylation and transcription sequencing (scNMT-Seq)
Isolate single cell
Lyse and GpC methylase labelling
AA(A)nSingle cell
polyA RNA
DNA methylation
Chromatin accessibility
Transcriptome
DNA methylation
Chromatin accessibility
mRNA
DNA T T T T T T T T T TAAAAAAA
Streptavidin magnetic bead with mRNA capture primer
Methylated CpG indicates accessible DNA
Isolate mRNA
T T T T T T T T T TAAAAAAA On-bead transcriptome
ampli�cation with Smart-seq2
Whole genome bisul�te sequencing with scBS-seq
scNMT-seq
Biotin
6 rounds of random priming with biotinylated adapters
Methylated GpC dinucleotides mark the absence of nucleosomes
Capture �rst strand on Streptavi-din-coated magnetic beads
Streptavidin
2nd random primer
Amplify and sequence
Methylated CpGdinucleotides
Methylated GpCdinucleotides
GpC methyl-transferaseLyse
Chromatin
NOMe-Seq PBAT
Bisul�te conversion
Chromatin overall omic-scale landscape sequencing (scCOOL-Seq)
Single cell
DNA methylation
CNV and ploidy
Chromatin accessibilityand nucleosome position
scCool-Seq
Sort cells to microplate wells Hypotonic lysis
Physical separation
SupernatantLibrary prep
Nuclei
Total RNA
From cellular mRNA
From genomic DNA
AntibodySurface antigen
Microwell
Magnetic microbeadDNA DNA
Simultaneous isolation of genomic DNA & total RNA from single-cells (SIDR)
Bead-binding prior to cell lysis reduces the number of cell-surface proteins that are solubilized
Hypotonic lysis releases cytoplasmic material but preserves the integrity of the nuclear membrane
Nuclei remain encapsulated by semi-per-meabilized cell membranes and can be isolated magnetically”
cDNA libraries are ready for sequencing
AA(A)nNon-polyA RNA
PolyA RNA
SIDRSingle cell
Crosslinked chromatin
dsDNA
Tagmented DNA
Hi-C
Restriction digest
Single cell
Fixed cell
Single-cell chromatin conformation capture method with multiplex end-tagging ampli�cation (Dip-C)
DNA close to each other in proximity are digested from chromatin and ligated to each other using DNA ligase. The removal of all biotin-pull-downsteps increases e�ciency
20 di�erent sequences of META barcodes reduce the amount of DNA lost due to fragments having the same sequencing tags to 1/20 of input DNA. PCR-based seq adapter addition also reduce arti�cial chimeras
Sophisticated algorithm can also identify chromosomal haplotypes linked by contact
META transposome
TransposaseSeq adapters
META PCR primers
META barcodesDNA
META PCR primer20 bp META barcode
DNA ligase
Lyse cell
Amplify with META primers
Dip-C
For Research Use Only. Not for use in diagnostic procedures.
© 2020 Illumina, Inc. All rights reserved. Illumina, Inc. • 5200 Illumina Way, San Diego, CA 92122 USA • 1.800.809.4566 toll-free • 1.858.202.4566 tel • [email protected] • illumina.comIllumina, HiSeq, MiSeq, MiniSeq, Nextera, NextSeq, TruSeq, the pumpkin orange color, and the Genetic Energy streaming bases design are trademarks or registered trademarks of Illumina, Inc. All other brands and names contained herein are the property of their respective owners. Pub. No. 770-2020-002-A QB9466. Current as of 14 April 2020.
This poster was compiled by the Illumina Scienti�c A�airs. Additional information, the latest version of the poster, and a comprehensive list of *seq methods, are available at http://www.illumina.com/libraryprepmethods. Please contact Scienti�c A�airs with any questions, comments, or suggestions.
ReferencesAct-Seq Wu Y. E. et al. (2017) Neuron 96(2): 313-329CEL-Seq Hashimshony T. et al. (2012) Cell Rep 2: 666-673CirSeq Acevedo A. et al. (2014) Nature 505: 686-690CITE-Seq Stoeckius M., et al. (2017) Nat Methods 14(9): 865-868CLaP Binan L. et al. (2016) Nat Commun 7: 11636CRISPR-UMI Michlits G. et al. (2017) Nat Methods 14(12): 1191-1197CROP-Seq Datlinger P. et al. (2017) Nat Methods 14(3): 297-301CytoSeq Fan H. C. et al. (2015) Science 347: 1258367Digital RNA Shiroguchi K. et al. (2012) Proc Natl Acad Sci USA
109:1347-1352Dip-C Tan L., et al. (2018) Science 361(6405): 924-928Div-Seq Habib N. et al. (2016) Science 353(6302): 925-928DP-Seq Bhargava V. et al. (2013) Sci Rep 3: 1740
DroNC-seq Habib N. et al. (2017) Nat Methods 14(10): 955-958Drop-Seq Macosko E. Z. et al. (2015) Cell 161: 1202-1214DR-Seq Dey S. S. et al. (2015) Nat Biotechnol 33: 285-9Drop-ChIP Rotem A. et al. (2015) Nat Biotechnol 33: 1165-72Duplex-Seq Schmitt M. W. et al. (2012) Proc Natl Acad Sci USA 109:
14508-14513ECCITE-seq Mimitou E. P. et al. (2019) Nat Methods 16(5): 409-412FREQ-Seq Chubiz L. M. et al. (2012) PLoS One 7: e47959FRISCR Thomsen E. R. et al. (2016) Nat Methods 13: 87-93G&T-seq Macaulay I. C. et al. (2015) Nat Methods 12: 519-522HiRes-Seq Imashimizu M. et al. (2013) Nucleic Acids Res 41:
9090-9104Hi-SCL Rotem A. et al. (2015) PLoS One 10: e0116328
IMS-MDA Seth-Smith H. M. et al. (2013) Nat Protoc 8: 2404-2412inDrop Klein A. M. et al. (2015) Cell 161: 1187-201LIANTI Chen C. et al. (2017) Science 356(6334): 189-194MALBAC Zong C. et al. (2012) Science 338: 1622-1626MARS-seq Jaitin D. A. et al. (2014) Science 343:776-9MATQ-seq Sheng K. et al. (2017) Nat Methods 14(3): 267-270MDA Dean F. B. et al. (2001) Genome Res 11: 1095-1099Microwell-seq Han X. et al. (2018) Cell 172(5): 1091-1107.e1017MIDAS Gole J. et al. (2013) Nat Biotechnol 31:1126-32MIPSTR Carlson K. D. et al. (2015) Genome Res 25: 750-761Mosaic-seq Han X. et al. (2018) Cell 172(5): 1091-1107 e1017MULTI-seq McGinnis C. S. et al. (2019) Nat Methods 16(7): 619-626NanoCAGE Plessy C. et al. (2010) Nat Methods 7: 528-534
NanogridSNRS Gao R. et al. (2017) Nat Commun 8(1): 228nuc-seq Wang Y. et al. (2014) Nature 512: 155-160Nuc-Seq/SNES Leung M. L. et al. (2015) Genome Biology 16(1): 55OS-Seq Myllykangas S. et al. (2011) Nat Biotechnol 29: 1024-1027PAIR Bell T. J. et al. (2015) Methods Mol Biol 1324: 457-68Quartz-Seq Sasagawa Y. et al. (2013) Genome Biol 14: R31Quartz-Seq2 Sasagawa Y. et al. (2018) Genome Biology 19(1): 29RamDA-seq Hayashi T. et al. (2018) Nature Communications 9(1): 619RNAtag-Seq Shishkin A. A. et al. (2015) Nat Methods 12: 323-325Safe-SeqS Kinde I. et al. (2011) Proc Natl Acad Sci USA 108: 9530-5scABA-seq Mooijman D. et al. (2016) Nature Biotechnology 34: 852scATAC-seq Buenrostro J. D. et al. (2015) Nature 523: 486-490 (Microfluidics)
scATAC-Seq Cusanovich D. A. et al. (2015) Science 348: 910-4 (Cell Index)scChip-seq Rotem A. et al. (2015) Nat Biotechnol 33: 1165-72scCool-seq Li L. et al. (2018) Nature Cell Biology 20(7): 847-858sciHi-C Ramani V. et al. (2017) Nature Methods 14: 263sci-CAR Cao J. et al. (2018) Science 361(6409): 1380sci-DNA-seq Rosenberg A. B. et al. (2018) Science 360: 176-182sci-MET Mulqueen R. M. et al. (2018) Nature Biotechnology 36: 428sci-RNA-seq Cao J. et al. (2017) Science 357(6352): 661SCMDA Dong X. et al. (2017) Nature Methods 14: 491scM&T-seq Angermueller C. et al. (2016) Nature Methods 13: 229scNMT-seq Clark S. J. et al. (2018) Nature Communications 9(1): 781scRC-Seq Upton K. R. et al. (2015) Cell 161: 228-39scRNA-seq Tang F. et al. (2009) Nat Methods 6: 377-82
SCRB-Seq Soumillon M. et al. (2014) bioRxiv: 003236scTHS-seq Lake B. B. et al. (2018) Nature Biotechnology 36(1): 70-80scTrio-seq Hou Y. et al. (2016) Cell Res 26: 304-19scTrio-seq2 Bian S. et al. (2018) Science 362(6418): 1060Seq-Well Gierahn T. M., et al. (2017). Nat Methods 14(4): 395-398SIDR Han K. Y. et al. (2018) Genome Research 28(1): 75-87SINC-seq Abdelmoez M. N. et al. (2018) Genome Biology 19(1): 66Smart-Seq Ramskold D. et al. (2012) Nat Biotechnol 30: 777-782Smart-seq2 Picelli S. et al. (2013) Nat Methods 10: 1096-1098vSMDB Lan F. et al. (2016) Nat Commun 7: 11784smMIP Hiatt J. B. et al. (2013) Genome Res 23: 843-854snDrop-seq Lake B. B. et al. (2018) Nature Biotechnology 36(1): 70-80SNES Leung M. L. et al. (2015) Genome Biol 16: 55
snmC-Seq Luo C. et al. (2017) Science 357(6351): 600snRNA-seq Grindberg R. V. et al. (2013) Proc Natl Acad Sci USA 110:
19802-7SPLiT-seq Rosenberg A. B. et al. (2018) Science 360(6385): 176STRT Islam S. et al. (2011) Genome Res 21: 1160-1167SUPeR-seq Fan X. et al. (2015) Genome Biol 16: 148TCR Chain Pairing Turchaninova M. A. et al. (2013) Eur J Immunol 43:
2507-2515TCR-LA-MC-PCR Ruggiero E. et al. (2015) Nat Commun 6: 8081TIVA Lovatt D. et al. (2014) Nat Methods 11: 190-196TSCS Casasent A. K. et al. (2018) Cell 172(1): 205-217.e212UMI Method Kivioja T. et al. (2012) Nat Methods 9: 72-74viscRNA-seq Zanini F. et al. (2018) Elife 7: e32942
Sequencing by Synthesis
TruSeq™ PCR Free
Double-stranded DNA
FractionateSize select
A-overhang
End repairPhosphorylate
PP
A
AP
P
T
P
P5
P7
IndexT
P
P5
P7
Index Adapter ligation
P5
P5P7Index
P7
Index
Add Adapters
Product ready for cluster generation
TruSeq™ NanoDouble-stranded DNA
FractionateSize select
A-overhang
End repairPhosphorylate
PP
A
AP
P
T
P
P5
P7
Index 1Index 2
Index 2Index 1
T
P
P5
P7
Adapter ligation
Denature and amplify
Add Adapters
P5
P7
Index 1Index 2
Index 2Index 1
P5
P7
P5 P7Index 1Index 2
Double-stranded DNA
Product ready for cluster generation
TruSeq™ Small RNA
3’5’ Small RNA fragment
Ligate adapters
Add primer
Reverse transcription
Denature and amplify
5’ Adapter 3’ Adapter
P7
Index 1
P5
P5 P7Index
Product ready for cluster generation
AATTCGC
AATTCGC
AATTCGC
AATTCGC
AATTCGC
Synthesize second strand
The second read is sequenced
Sequence Index2
AATTCGC
Deblock P5 primer and add unlabeled bases
Read 2 primer
The forward-strand is cleaved and washed away
AATTCGC
AATTCGC
AATTCGC
AATTCGC
AATTCGC
AATTCGC
Adapter hybrid-izes to flowcell
Reverse strand synthesis
Reverse strand
Forward strand
Remove forward strand
Fold over and hybridize to second primer
Synthesize second strand
The reverse strand is cleaved and washed away
With each cycle, four fluores-cently tagged nucleotides compete for addition to the growing chain. Only one is incorporated based on the sequence of the template.
The read product is washed away
Thousands of molecules are amplified in parallel
Reverse strand
Forward strand
Bridge amplification
Sequence primer
Fold over and hybridize to first primer
Fold over and hybridize to first primer
Sequence Index1
Index 1 primer
The read product is washed away
TruSeq™ RNA Exome
Elute
Target
TargetP5 P7
Index 1Index 2 Product ready for cluster generation
Pool stranded RNA-Seq libraries
Biotinylated target probe
Hybridize probes to targets
Capture on streptavidin magnetic beads
TruSeq™ Targeted RNA ExpressionTarget
ULSO DLSO
Total RNA
cDNA
Hybridization
P7
Index 1
P5
P5 P7
5’ P
5’ P
Index 2
Target Index 1Index 2 Product ready for cluster generation
Add custom primers
Denature and amplify
Extension-Ligation
Nextera™ Library Preparation
Transposase
DNA
~300bp
Tagmentation
Ampli�cation
P5 P7Index 1Index 2
P5
Index 2
Index 1
P7
Product ready for cluster generation
Nextera™ Mate Pair
Adapter ligation
Isolate biotinylatedfragment
Denature and amplify
P5 P7
Transposase
DNA
Tagmentation
Circularize
R R R RBiotinylated junction adapter
R
R
R
R
R
R
Fragment
R
R
R
R
P5
P7 P5
P7R
R
Product ready for cluster generation
Nextera™ Rapid Capture
Elute
Target
TargetP5 P7
Index 1Index 2 Product ready for cluster generation
Denatured and pooled fragments from Nextera library
Capture onstreptavidin magnetic beads
Hybridize probes to targets
Biotinylated target probe
AmpliSeq™ for Illumina
Remove primer sequences
Add sequencing primers
Index 2Index 1
P5
P7
PCR
Index 1P7
Index 2P5 Product ready for
cluster generation
DNA/cDNA
DNA RNA5’ 3’
PCR
Reversetranscribe
Ligate adaptors
TruSeq™ RNA
Total RNA
T T T T TAAAAA polyA select
Fragment
Random hexamer
First and second strand synthesis
5’ 3’
TruSeq™ Stranded RNARNA/mRNARandom primer
Create cDNA
Create second strand cDNA
End repairPhosphorylateA-overhang
Adaptor ligation
Denature and amplify
cDNA
dUTP + dCTP + dATP + dGTP
dT TP + dCTP + dATP + dGTP
P5 P7
P5 P7Index 1Index 2
U U U UUUUUUUUSense strand
A
AP
PU U U UUUUUUUSense strand
P7
Index 1
P5Index 2
U U U UUUUUUU Index 2
P5
Index 1
P7
Sense strand
P5P7U U U UUUUUUU
Sense strand Block polymerase
Product ready for cluster generation
5’ 3’ AAAAA
SureCell™ WTA 3’
Ampli�cation
3’ enrichment and sample indexing
Product ready for cluster generation
Direct cDNA Nextera tagmentation
Cell lysismRNA hybridization
Single cells encapsu-lated in droplets
cDNA synthesis and barcoding
Barcoded beads
UMI Barcode Read1T T T T TAAAAA
mRNAUMI Barcode Read1
T T T T T
mRNAAAAAA
UMI Barcode Read1T T T T T
mRNAAAAAA
Index
P7
P5
IndexP7 P5
UMI Barcode Read1
cDNA