chip-seq kit for histones (high sensitivity) · 2017. 11. 18. · 9. sonicator tubes 10. gloves 11....

Boster Biological Technology 3942 Valley Ave., Ste. B, Pleasanton, CA 94566 Phone: 888-466-3604 Fax: 925-215-2184 Email: [email protected] Web: www.bosterbio.com

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ChIP-seq Kit for Histones (High Sensitivity)

An all-in-one kit for ChIP assay prior to NGS, validated for GAIIx (Illumina) and PGM (Ion Torrent)

Catalog numbers CK1001-24 (24 reactions) CK1001-100 (100 reactions) CK1001-10 (10 reactions)

Applications ChIP-qPCR, ChIP-seq Pack Size 1 kit (24 components) Sample Type Cultured cells, tissues (fresh or frozen)

Number of Chromatins Prepared 4 (24 reactions) 17 (100 reactions) 2 (10 reactions)

Kit Components

Quantity Quantity Quantity

No. Component (24 rxns) (100 rxns) (10 rxns) Storage

1 ChIP-seq grade H3K4me3 antibody (positive IP control), 1 µg/µL 10 µg 40 µg 10 µg -20˚C (1 year)*

2 ChIP-seq grade GAPDH TSS primer pair, human (+ve control) 100 µL 500 µL 25 µL -20˚C (1 year)*

3 ChIP-seq grade myoglobin exon 2 primer pair, human (-ve control) 100 µL 500 µL 25 µL -20˚C (1 year)*

4 Rabbit IgG antibody (negative IP control), 1 µg/µL 10 µg 40 µg 10 µg -20˚C (1 year)*

5 5% BSA (DNA free) 175 µL 720 µL 70 µL -20˚C (1 year)*

6 Carrier 80 µL 320 µL 55 µL -20˚C (1 year)*

7 Glycine 480 µL 1.9 mL 175 µL 4˚C (1 year)

8 200X protease inhibitor cocktail 90 µL 365 µL 30 µL -20˚C (1 year)*

9 Magnetic beads (Protein A-coated) 580 µL 2.2 mL 240 µL 4˚C (1 year)**

10 Magnetic beads (High DNA recovery) 385 µL 1.6 mL 400 µL 4˚C (1 year)**

11 ChIP-seq grade water 10 mL 40 mL 4 mL 4˚C (1 year)

12 Lysis buffer B 44 mL 190 mL 15 mL 4˚C (1 year)

13 Lysis buffer C 44 mL 190 mL 15 mL 4˚C (1 year)

14 Shearing buffer B 8 mL 34 mL 2 mL 4˚C (1 year)

15 5X ChIP buffer B 4 mL 11 mL 1.7 mL 4˚C (1 year)

16 Wash buffer A 10 mL 42 mL 4 mL 4˚C (1 year)

17 Wash buffer B 10 mL 42 mL 4 mL 4˚C (1 year)

18 Wash buffer C 10 mL 42 mL 4 mL 4˚C (1 year)

19 Wash buffer D 10 mL 42 mL 4 mL 4˚C (1 year)

20 Wash buffer 1 (w/o iso-propanol) 2 mL 8 mL 2 mL 4˚C (1 year)

21 Wash buffer 2 (w/o iso-propanol) 2 mL 8 mL 2 mL 4˚C (1 year)

22 Elution buffer A 4 mL 16 mL 6 mL 4˚C (1 year)

23 Elution buffer B 175 µL 720 µL 240 µL 4˚C (1 year)

24 Elution buffer C 2 mL 8 mL 4 mL 4˚C (1 year)

* Avoid repeated freeze/thaw ** Do not freeze


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Required But Not Provided

Reagents and Materials

1. Formaldehyde, 37%, Molecular Grade 2. PBS Buffer 3. 1M Sodium Butyrate 4. 100% Isopropanol 5. qPCR SYBR® Green Mastermix 6. ChIP-Seq Reagents (Illumina or Thermo Fisher) 7. Quant-IT dsDNA HS Assay kit (Thermo Fisher) 8. RNase/DNase-Free 1.5 mL Tubes 9. Sonicator Tubes 10. Gloves 11. Petri Dishes 12. Petri Dishes 13. Scalpel Blades Equipment

1. Magnetic Racks 2. Sonicator 3. Cell Counter 4. Rotating Wheel 5. Refrigerated Centrifuges 6. Qubit Fluorometer (Thermo Fisher) 7. Thermomixer 8. qPCR Cycler 9. Vortex 10. Tissue Homogenizer

ChIP-Seq Data Images

High consistency of the Boster ChIP-seq kit on

the PGM (Ion Torrent) and GAIIx (Illumina):

ChIP was performed on sheared chromatin

from 1 million HelaS3 cells using the ChIP-seq

kit and 1 µg of H3K4me3 positive control

antibody. Two different biological samples

have been analyzed using the two different

sequencers. The expected ChIP-seq profile for

H3K4me3 on the GAPDH promoter region has

been obtained. (A) A several hundred bp along

chr12 with high similarity of read distribution is

seen despite the radically different

sequencers. (B) A close capture focusing on

the GAPDH that shows that even the peak

structure is similar.


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Assay Principle

Chromatin immunoprecipitation (ChIP) is a technique to analyze the association of proteins with specific genomic regions in intact cells. The different steps of the ChIP assay are: 1) Cell fixation (cross-linking) 2) Chromatin shearing 3) Immunoprecipitation 4) Reverse cross-linking 5) DNA purification 6) Analysis of the immunoprecipitated DNA In ChIP, living cells are first fixed with a reversible cross-linking agent to stabilize protein-DNA interactions. The most widely

used reagent to fix cells is formaldehyde which generates covalent bonds between amino or imino groups of proteins and

nucleic acids. Formaldehyde treatment crosslinks both DNA-protein as well as protein-protein complexes. Following cross-

linking, chromatin needs to be sheared very efficiently into homogeneous small fragments that can subsequently be used in

immunoprecipitation. After fragmentation, the sheared chromatin is precipitated with a specific antibody directed against the

protein of interest. The chromatin-antibody complex is isolated using magnetic beads. Finally, the precipitated DNA fragments

Efficient and easy chromatin shearing using the Boster ChIP-

seq kit for Histones. Samples (Chromatin from 1 million of

Hela cells) were gently vortexed before and after performing

each sonication round followed by a short centrifugation at

4°C to recover the sample volume at the bottom of the tube.

The sheared chromatin was then decross-linked as described

in the kit manual and analyzed by agarose gel

electrophoresis.

qPCR-based ChIP validation using Boster’s ChIP-seq grade

H3K4me3 antibody, isotype control and validated primers:

Specific enrichment on positive loci (GAPDH, EIF4A2, c-fos

promoter regions) comparing to no enrichment on negative

loci (TSH2B promoter region and Myoglobin exon 2) was

detected by qPCR.


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are released from the antibody and analyzed. Enrichment of specific sequences in the precipitated DNA indicates that these

sequences were associated with the protein of interest in vivo. Analysis of specific regions can be performed by quantitative

polymerase chain reaction (qPCR). In recent years, ChIP combined with high-throughput Next-Generation sequencing (NGS)

known as ChIP-seq has become the gold standard for whole-genome mapping of protein-DNA interactions.

Notice Before Starting

1. Cell Number (For Cultured Cells): This protocol has been optimized for ChIP on 1 million cells in a 300 µL ChIP reaction.

It is possible to use more cells. However, for optimal performance, we recommend performing separate ChIPs and pool

the immunoprecipitated DNA before purification.

2. Tissue Amount (For Fresh or Frozen Tissues): This protocol has been optimized for ChIP from fresh or frozen

mammalian tissues. The chromatin is prepared from 30-40 mg of tissue allowing up to 18 ChIP samples (about 1.5 to 2 mg

of tissue per IP). However, the exact amount of tissue needed for ChIP-seq may vary depending on protein abundance,

antibody affinity, etc. and should be determined for each tissue type. We recommend performing a pilot experiment.

3. Shearing Optimization and Sheared Chromatin Analysis: Before starting the ChIP, the chromatin should be sheared

into fragments of 100 to 600 bp. You may use the Bioruptor® (Diagenode) with a maximum shearing volume of 300 µL in

a 1.5 mL TPX microtube (Diagenode). The shearing conditions mentioned in the protocol are adequate for a variety of cell

types. However, given that cell types are different, we recommend optimizing sonication conditions for each cell type before

processing large quantities of cells or samples. It is important to perform an initial sonication time course experiment to

evaluate the extent of chromatin fragmentation.

4. Magnetic Beads: This kit includes the Protein A-coated magnetic beads. Make sure the beads do not dry during the

procedure as this will result in reduced performance. Keep the beads homogenously in suspension at all times when

pipetting. Variation in the amount of beads will lead to lower reproducibility. Do not freeze the beads. The amount of beads

needed per IP depends on the amount of antibody used. This protocol uses 20 µL of beads. The binding capacity of this

amount is approximately 5 µg of antibody. We recommend to use is 1 to 2 µg of ChIP-seq grade antibody per IP reaction.

Therefore, you can reduce the amount of beads accordingly.

5. Negative and Positive IP Controls: The kit contains a negative (IgG) and a positive (H3K4me3) control antibody. We

recommend including one IgG negative IP control in each series of ChIP reactions. We also recommend using the positive

control ChIP-seq grade H3K4me3 antibody at least once. The kit also contains qPCR primer pairs for amplification of a

positive and negative control target for H3K4me3 (GAPDH-TSS and Myoglobin exon 2, respectively).

6. Quantification: The post-ChIP immunoprecipitated DNA concentration can be determined with a highly sensitive method

such as the 'Quant-IT dsDNA HS assay kit’ on the Qubit system (Thermo Fisher). PicoGreen is also suitable but UV

spectrophotometric methods (e.g. NanoDrop) are usually not sufficiently sensitive. In most cases it is sufficient to use

approximately 10% of the immunoprecipitated material for quantification. The expected DNA yield depends on different

factors such as the cell type, antibody quality and antibody target. The expected DNA yield obtained with the positive control

H3K4me3 antibody on 1 million HeLa cells is about 10 ng.

7. Quantitative PCR: Before sequencing the samples, we recommend analyzing the immunoprecipitated DNA by qPCR

using at least one positive and one negative control targets. This kit contains a positive and negative control primer pair

which can be used for the H3K4me3 positive control antibody in SYBR® Green qPCR assay. Use your own method of

choice for analyzing the appropriate control targets for your antibodies of interest. In order to have sufficient DNA left for

sequencing, we recommend not using more than 10% of the total immunoprecipitated DNA for qPCR. You can dilute the

DNA (1/10 or more) to perform sufficient PCR reactions. PCR reactions should be performed at least in duplicate although

performing them in triplicate is recommended to be able to identify potential outliers.

8. Quantitative PCR Data Interpretation: The efficiency of chromatin immunoprecipitation of particular genomic loci can be

expressed as the recovery of that locus calculated as the percentage of the input (the relative amount of immunoprecipitated

DNA compared to input DNA). If the amount used for the input was 1% of the amount used for ChIP, the recovery can be

calculated as follows: % recovery = 2^(Ctinput – Ctsample) where Ctsample and Ctinput are the threshold cycles from the

exponential phase of the qPCR for the immunoprecipitated DNA sample and input, respectively. This equation assumes

that the PCR is 100% efficient (amplification efficiency = 2). The real amplification efficiency, if known, should be used for


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accurate results. For the positive control antibody (H3K4me3), the recovery of the positive control target (GAPDH TSS

locus) is expected to be between 10% and 20% although this depends on the cell type used. The recovery of the negative

control target (Myoglobin exon 2 locus) should be below 1%.

Assay Procedure (24 and 100 Reactions)

1. Cell Collection and DNA-Protein Cross-Linking (For Cultured Cells)

The protocol below is intended for adherent cells. For suspension cells, collect them by centrifugation (start with step

stated with a * below).

Pre-warm PBS, culture medium and trypsin-EDTA at 37°C.

Remove the medium and rinse the cells with pre-warmed PBS (10 mL for a 75 cm2 culture flask). Gently shake the

flask for 2 min.

Remove the PBS and add sterile trypsin-EDTA to the culture flask to detach adherent cells from the bottom. The table

below shows the required amount of trypsin for different numbers of cells. Gently shake the culture flask for 1-2 min

or until the cells start to detach. The time needed may depend on the cell type.

Number of Cells Trypsin-EDTA Volume

3 x 106 1 mL

1 x 107 3 mL

5 x 107 15 mL

Note: Do not continue trypsin treatment longer than necessary as prolonged treatment with trypsin may damage the

cells. Regularly check if the cells start to detach.

Immediately add fresh culture medium to the cells when they are detached (refer to the table below for volume). This

will inactivate trypsin. Transfer cell suspension to a 50 mL tube.

Number of Cells Culture Medium Volume

3 x 106 2 mL

1 x 107 6 mL

5 x 107 30 mL

Rinse the flask by adding 10 mL of PBS. Add this volume to your 50 mL tubes containing cells from the above bullet.

* Centrifuge for 5 min at 1,600 rpm and 4°C and remove the supernatant.

Resuspend the cells in 20 mL of PBS and count them.

Collect the cells by centrifugation for 5 min at 1,600 rpm and 4°C.

Resuspend the cells in PBS to obtain a concentration of up to 10 million cells per 500 µL of PBS. If desired, the cell

concentration can be decreased to 1 million per 500 µL of PBS.

Label 1.5 mL tubes and aliquot 500 µL of cell suspension in each tube.

Add 13.5 µL of formaldehyde 37% to each tube containing 500 µL of cell suspension. Mix by gentle vortexing and

incubate for 8 min at room temperature to allow fixation to take place.

Add 57 µL of glycine to the cells to stop the fixation. Mix by gentle vortexing and incubate for 5 min at room

temperature. Keep the cells on ice from this point onwards.

Collect the cells by centrifugation at 1,600 rpm for 5 min and 4°C. Discard the supernatant without disturbing the cell

pellet.

Wash the cells 2X with 1 mL of cold PBS.

Proceed to 3: Cell Lysis and Chromatin Shearing (For Cultured Cells).

(To be continued on next page)


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2. Tissue Disaggregation and DNA-Protein Cross-Linking (For Fresh and Frozen Tissue)

Weigh 30-40 mg of fresh or frozen tissue in a petri dish. Keep samples on ice at all times and minimize the time of

manipulation to prevent sample degradation.

Chop tissue into small pieces (between 1-3 mm3) using a scalpel blade.

Add 1 mL of ice-cold PBS with protease inhibitor cocktail and disaggregate the tissue using a dounce homogenizer

(loose pestle) to get a homogeneous suspension.

Transfer the tissue suspension into a 1.5 mL tube and centrifuge at 1,300 rpm for 5 min at 4°C. Gently discard the

supernatant and keep the pellet.

Resuspend the pellet in 1 mL of PBS containing 1% of formaldehyde at room temperature.

Rotate tube for 8-10 min at room temperature.

Note: The fixation time might require an additional optimization. In general, histone marks require shorter fixation (8

min) than transcriptional factors (10-15 min). Stronger fixation may lead to chromatin resistant to sonication.

Stop the cross-linking reaction by adding 100 µL of glycine. Continue to rotate at room temperature for 5 min.

Centrifuge samples at low speed (1,300 rpm) at 4°C.

Wash the pellet with ice-cold PBS. Aspirate the supernatant and resuspend the pellet in 1 mL of ice-cold PBS plus

protease inhibitors.

Centrifuge at low speed (1,300 rpm) at 4°C and discard the supernatant.

Repeat the washing 1X.

Proceed to 4: Cell Lysis and Chromatin Shearing (Derived from Tissue Samples).

3. Cell Lysis and Chromatin Shearing (For Cultured Cells)

Add 1 mL of ice-cold lysis buffer C to the 1.5 mL tube containing 10 million cells. Resuspend the cells by pipetting up

and down several times and transfer them to a 15 mL tube. Add 9 mL of lysis buffer C and incubate for 10 min at 4°C

with gentle mixing.

Note: If the starting amount of cells was less than 10 million, scale down accordingly (e.g. Use a total of 5 mL lysis

buffer C for 5 million cells).

Pellet the cells by centrifugation at 1,600 rpm for 5 min and 4°C and discard the supernatant.

Add 1 mL of ice-cold lysis buffer B and resuspend the cells by pipetting up and down several times. Add another 9

mL of lysis buffer B and incubate for 10 min at 4°C with gentle mixing. Scale down accordingly when using less than

10 million cells.

Pellet the cells again by centrifugation for 5 min at 1,600 rpm (500 x g) and 4°C and discard supernatant.

Add 200X protease inhibitor cocktail to shearing buffer B. Prepare 1 mL of complete shearing buffer per tube of 10

million cells. Keep on ice.

Add 1 mL of complete shearing buffer B to 10 million cells. Resuspend the cells by pipetting up and down several

times. The final cell concentration should be 1 million cells per 100 µL shearing buffer B.

Split into aliquots of 100 to 300 µL and transfer the cell suspension to 1.5 mL sonication tubes.

Incubate on ice for 10 min. Vortex and spin down the samples.

Split the samples into 300 µL aliquots in 1.5 mL sonication tubes and incubate on ice for 10 min.

Centrifuge at 13,000 rpm (16,000 x g) for 10 min and collect the supernatant which contains the sheared chromatin.

Use the chromatin immediately in immunoprecipitation or store it at -80°C for up to 2 months.

Proceed to 5: Magnetic Immunoprecipitation.

4. Cell Lysis and Chromatin Shearing (Derived from Tissue Samples)

Add 10 mL of ice-cold lysis buffer C to the pellet corresponding to 30-40 mg of tissue.

Resuspend the pellet by pipetting up and down and incubate for 10 min at 4°C with gentle mixing.

Centrifuge for 5 min at 1,300 rpm at 4°C and discard the supernatant.


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Add 10 mL of ice-cold lysis buffer B to the pellet. Resuspend the pellet by pipetting up and down and incubate for 10

min at 4°C with gentle mixing.


Resuspend the pellet in 1.8 mL of shearing buffer B containing protease inhibitors cocktail and homogenize using a

dounce homogenizer (tight pestle).


Shear chromatin by sonication (Optimization on shearing conditions, e.g. number of runs, number of cycles per run,

may be required, depending on the sonicator and cell types used). Vortexing is not required between runs.

Transfer samples to new 1.5 mL tubes and centrifuge at 13,000 rpm for 10 min.

Collect the supernatant which contains the sheared chromatin.

Store the chromatin at -80°C for up to 2 months for further use in the immunoprecipitation.

5. Magnetic Immunoprecipitation

Note: This protocol has been optimized for 1 million cells per ChIP. Although it is possible to use more cells, we

recommend performing separate ChIP reactions and pool the samples before purification of the DNA.

Determine the total number of IPs in the experiment. We recommend to include one negative control in each

experiment (IP with the IgG negative control). Take the required amount of protein A-coated magnetic beads (20

µL/IP).

Dilute the 5X ChIP buffer B with ChIP-seq grade water to obtain 1X ChIP buffer B. The total amount of 1X ChIP buffer

B needed is 9X the volume of beads required for the experiment. Place the diluted ChIP buffer on ice.

Wash the beads 4X with 2X volume of ice-cold 1X ChIP buffer B. To wash the beads, add 1X ChIP buffer, resuspend

the beads by pipetting up and down several times and place the tubes in a suitable magnetic rack for 1.5-mL tubes.

Wait for one min to allow the beads to be captured by the magnet and remove the supernatant.

Repeat the above 3X. Alternatively, centrifuge the tubes for 5 min at 1,300 rpm, discard the supernatant and keep

the bead pellet.

After the last wash, resuspend the beads in the original volume of 1X ChIP Buffer.

Prepare the ChIP reaction mix according to the table below.

No. of IPs

5% BSA

200X Protease Inhibitor Cocktail

5X ChIP Buffer B

Sheared Chromatin (1x1010 cells)

Magnetic Beads

ChIP-seq Grade Water

Antibody

1 6 µL 1.5 µL 56 µL 100 µL 20 µL (116.5 – V) µL V µL

2 12 µL 3.0 µL 112 µL 200 µL 40 µL (233 – V) µL V µL




Note: If required, sodium butyrate NaBu, a potent deacetylase inhibitor, (20 mM final concentration) or other inhibitors

can also be added.

Add the antibody to the reaction mix. Use 2 µL of the rabbit IgG control antibody for the negative control IP. If a

positive control IP is included in the experiment, use 1 µL of the H3K4me3 ChIP-grade control antibody.

When preparing the reaction mix, place 1 µL of the sheared chromatin aside to be used as an input the next day.

Incubate the tubes overnight at 4°C under constant rotation at 40 rpm.

The next morning, after the overnight incubation, briefly spin the tubes and place them in a magnetic rack. Wait for

one min and remove the supernatant.

Wash the beads by adding 350 µL of wash buffer A, gently shaking the tubes to resuspend the beads, and incubating

for 5 min at 4°C under constant rotation at 40 rpm.

Repeat the wash as described above 1X with wash buffers B, C and D, respectively.

(To be continued on next page)


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6. Elution, Decross-Linking and DNA Isolation

After removing the last wash buffer, add 100 µL of elution buffer A to the beads and incubate for 30 min under constant

rotation at room temperature. Resuspend the beads pellet and transfer it into a new 200 µL tube.

Briefly spin the tubes and place them in a magnetic rack.

Transfer the supernatant to a new tube and add 4 µL of elution buffer B. Also add 99 µL elution buffer A and 4 µL of

elution buffer B to the 1 µL input sample kept aside the day before.

Incubate for 4 hours in a thermomixer at 1,300 rpm and 65°C. If required, the incubation at 65°C can be performed

overnight.

Pool samples if necessary.

Note: Up to two samples can be easily pooled. If more than two samples need to be pooled, process each sample

purification individually, pool final eluates at the end of the magnetic bead purification and concentrate.

Add 2 µL of carrier to each IP and input sample.

Vortex briefly and perform a short spin.

Add 100 µL of 100% isopropanol to each IP and input sample.


Note: Following the addition of isopropanol the solution may become cloudy. This is not detrimental to your experiment

and will not influence the quality or quantity of your purified DNA.

Resuspend the high-DNA recovery magnetic beads and transfer 10 µL to each IP and input sample (Final volume

should be 116 µL per reaction).

Note: Keep the beads in liquid suspension during storage at 4°C and at all handling steps, as drying will result in

reduced performance.

Incubate IP and input samples for 10 minutes at room temperature on a rotating wheel (40 rpm).

For 24 reactions, prepare the wash buffer 1 containing 50% isopropanol by mixing 2 mL wash buffer 1 w/o

isopropanol and 2 mL isopropanol (100%). Never leave the bottle open to avoid evaporation. For 100

reactions, mix 8 mL wash buffer 1 w/o isopropanol with 8 mL isopropanol (100%).

Briefly spin the tubes, place them in a suitable magnetic rack for 0.2-mL tubes, wait 1 min and discard the buffer.

Add 100 µL wash buffer 1 per tube. Close the tubes and vortex well until the beads pellet is completely broken. In

order to avoid the beads pellet to be too difficult to break down, do not let the beads for too long on the magnet,

incubate for 30 seconds at room temperature on a rotating wheel (40 rpm).

Note: Do not disturb the captured beads attached to the tube wall. Always briefly spin the tubes to bring down liquid

caught in the lid prior to positioning into the magnetic rack.

For 24 reactions, prepare the wash buffer 2 containing 50% isopropanol as follows by mixing 2 mL wash buffer 2 w/o

isopropanol and 2 mL isopropanol (100%). Never leave the bottle open to avoid evaporation. For 100 reactions, mix

8 mL wash buffer 2 w/o isopropanol with 8 mL isopropanol (100%).

Briefly spin the tubes, place in a suitable magnetic rack for 1.5-mL tubes, wait 1 min and discard the buffer.

Add 100 µL wash buffer 2 per tube. Close the tubes and vortex well until the beads pellet is completely broken. In

order to avoid the beads pellet to be too difficult to break down, do not let the beads for too long on the magnet,

incubate for 30 seconds at room temperature on a rotating wheel (40 rpm).



Briefly spin the tubes and place them into a 0.2-mL magnetic rack, wait 1 min and discard the buffer.

Spin the tubes again and place them on the magnetic rack. Discard the remaining Wash buffer 2 if necessary.

Resuspend the beads pellet with 25 µL of elution buffer C. Incubate at room temperature for 15 minutes on a rotating

wheel (40 rpm).


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Spin the tubes and place them into a 1.5-mL magnetic rack, wait 1 min and transfer the supernatants into a new

labelled 1.5 mL tube and discard the beads

Place the DNA on ice and proceed to any desired downstream applications or store it at -20°C or -80°C until further

use.

Take 5 µL (10%) of immunoprecipitated DNA and determine the concentration with, for example, the Quant-iT High

Sensitivity dsDNA Assay Kit and Qubit fluorometer.

Store the DNA at -20°C until you are ready to analyze it with qPCR or by high throughput sequencing.

Note: The elution buffer C is suitable for direct qPCR analysis, whole genome amplification, chip hybridization and

Next-Generation sequencing.

7. Quantitative PCR Analysis

Before sequencing the samples, we recommend analyzing the immunopreciptated DNA by qPCR using at least one

positive and one negative control target. The kit contains a positive (GAPDH SS) and negative (Myoglobin exon 2) control

primer pair which can be used for the positive control antibody provided in the kit (ChIP-seq grade H3K4me3 antibody) in

SYBR Green qPCR assay using the protocol described below. Use your own method of choice for analyzing the

appropriate control targets for your antibodies of interest.

Prepare the qPCR mix as follows (20 µL reaction volume using the provided control primer pairs):

10 µL of a 2X SYBR Green qPCR master mix

1 µL of primer mix

4 µL of water

5 µL immmunoprecitated or input DNA

Carry out the PCR with this sequence:

Denaturation step: 95°C (3 to 10 min)

40 cycles of 95°C (30 sec), 60°C (30 sec) and 72°C (30 sec).

Note: Check carefully supplier’s recommendations about Taq polymerase activation time. These PCR conditions may

require optimization depending on the type of Master Mix or qPCR system used.

8. ChIP-seq

This protocol has been optimized for ChIP-seq on an Illumina GAIIx and Ion Torrent next-generation sequencers.

However, other sequencing systems such as the Illumina HiSeq or the Thermo Fisher SOLiD systems can also be used.

Refer to their sequencing protocols for the generation of sequencing data.

Assay Procedure (10 Reactions)

1. Cell Collection and DNA-Protein Cross-Linking (For Cultured Cells)

The protocol below is intended for adherent cells. For suspension cells, collect them by centrifugation (start with step

stated with a * below).

Pre-warm PBS, culture medium and trypsin-EDTA at 37°C.

Remove the medium and rinse the cells with pre-warmed PBS (10 mL for a 75 cm2 culture flask). Gently shake the

flask for 2 min.

Remove the PBS and add sterile trypsin-EDTA to the culture flask to detach adherent cells from the bottom. The table

below shows the required amount of trypsin for different numbers of cells. Gently shake the culture flask for 1-2 min

or until the cells start to detach. The time needed may depend on the cell type.


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Number of Cells Trypsin-EDTA Volume

3 x 106 1 mL

1 x 107 3 mL

5 x 107 15 mL

Note: Do not continue trypsin treatment longer than necessary as prolonged treatment with trypsin may damage the

cells. Regularly check if the cells start to detach.

Immediately add fresh culture medium to the cells when they are detached (refer to the table below for volume). This

will inactivate trypsin. Transfer cell suspension to a 50 mL tube.

Number of Cells Culture Medium Volume

3 x 106 2 mL

1 x 107 6 mL

5 x 107 30 mL

Rinse the flask by adding 10 mL of PBS. Add this volume to your 50 mL tubes containing cells from the above bullet.

* Centrifuge for 5 min at 1,600 rpm and 4°C and remove the supernatant.

Resuspend the cells in 20 mL of PBS and count them.

Collect the cells by centrifugation for 5 min at 1,600 rpm and 4°C.

Resuspend the cells in PBS to obtain a concentration of up to 10 million cells per 500 µL of PBS. If desired, the cell

concentration can be decreased to 1 million per 500 µL of PBS.

Label 1.5 mL tubes and aliquot 500 µL of cell suspension in each tube.

Add 13.5 µL of formaldehyde 37% to each tube containing 500 µL of cell suspension. Mix by gentle vortexing and

incubate for 8 min at room temperature to allow fixation to take place.

Add 57 µL of glycine to the cells to stop the fixation. Mix by gentle vortexing and incubate for 5 min at room

temperature. Keep the cells on ice from this point onwards.

Collect the cells by centrifugation at 1,600 rpm for 5 min and 4°C. Discard the supernatant without disturbing the cell

pellet.

Wash the cells 2X with 1 mL of cold PBS.

Proceed to 3: Cell Lysis and Chromatin Shearing (For Cultured Cells).

2. Tissue Disaggregation and DNA-Protein Cross-Linking (For Fresh and Frozen Tissue)

Weigh 30-40 mg of fresh or frozen tissue in a petri dish. Keep samples on ice at all times and minimize the time of

manipulation to prevent sample degradation.

Chop tissue into small pieces (between 1-3 mm3) using a scalpel blade.

Add 1 mL of ice-cold PBS with protease inhibitor cocktail and disaggregate the tissue using a dounce homogenizer

(loose pestle) to get a homogeneous suspension.

Transfer the tissue suspension into a 1.5 mL tube and centrifuge at 1,300 rpm for 5 min at 4°C. Gently discard the

supernatant and keep the pellet.

Resuspend the pellet in 1 mL of PBS containing 1% of formaldehyde at room temperature.

Rotate tube for 8-10 min at room temperature.

Note: The fixation time might require an additional optimization. In general, histone marks require shorter fixation (8

min) than transcriptional factors (10-15 min). Stronger fixation may lead to chromatin resistant to sonication.

Stop the cross-linking reaction by adding 100 µL of glycine. Continue to rotate at room temperature for 5 min.

Centrifuge samples at low speed (1,300 rpm) at 4°C.

Wash the pellet with ice-cold PBS. Aspirate the supernatant and resuspend the pellet in 1 mL of ice-cold PBS plus

protease inhibitors.

Centrifuge at low speed (1,300 rpm) at 4°C and discard the supernatant.

Repeat the washing 1X.

Proceed to 4: Cell Lysis and Chromatin Shearing (Derived from Tissue Samples).


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11

3. Cell Lysis and Chromatin Shearing (For Cultured Cells)

Add 1 mL of ice-cold lysis buffer C to the 1.5 mL tube containing 10 million cells. Resuspend the cells by pipetting up

and down several times and transfer them to a 15 mL tube. Add 9 mL of lysis buffer C and incubate for 10 min at 4°C

with gentle mixing.

Note: If the starting amount of cells was less than 10 million, scale down accordingly (e.g. Use a total of 5 mL lysis

buffer C for 5 million cells).

Pellet the cells by centrifugation at 1,600 rpm for 5 min and 4°C and discard the supernatant.

Add 1 mL of ice-cold lysis buffer B and resuspend the cells by pipetting up and down several times. Add another 9

mL of lysis buffer B and incubate for 10 min at 4°C with gentle mixing. Scale down accordingly when using less than

10 million cells.

Pellet the cells again by centrifugation for 5 min at 1,600 rpm (500 x g) and 4°C and discard supernatant.

Add 200X protease inhibitor cocktail to shearing buffer B. Prepare 1 mL of complete shearing buffer per tube of 10

million cells. Keep on ice.

Add 1 mL of complete shearing buffer B to 10 million cells. Resuspend the cells by pipetting up and down several

times. The final cell concentration should be 1 million cells per 100 µL shearing buffer B.

Split into aliquots of 100 to 300 µL and transfer the cell suspension to 1.5 mL sonication tubes.

Incubate on ice for 10 min. Vortex and spin down the samples.


Centrifuge at 13,000 rpm (16,000 x g) for 10 min and collect the supernatant which contains the sheared chromatin.

Use the chromatin immediately in immunoprecipitation or store it at -80°C for up to 2 months.

Proceed to 5: Magnetic Immunoprecipitation.

4. Cell Lysis and Chromatin Shearing (Derived from Tissue Samples)

Add 10 mL of ice-cold lysis buffer C to the pellet corresponding to 30-40 mg of tissue.

Resuspend the pellet by pipetting up and down and incubate for 10 min at 4°C with gentle mixing.


Add 10 mL of ice-cold lysis buffer B to the pellet. Resuspend the pellet by pipetting up and down and incubate for 10

min at 4°C with gentle mixing.


Resuspend the pellet in 1.8 mL of shearing buffer B containing protease inhibitors cocktail and homogenize using a

dounce homogenizer (tight pestle).


Shear chromatin by sonication (Optimization on shearing conditions, e.g. number of runs, number of cycles per run,

may be required, depending on the sonicator and cell types used). Vortexing is not required between runs.

Transfer samples to new 1.5 mL tubes and centrifuge at 13,000 rpm for 10 min.

Collect the supernatant which contains the sheared chromatin.

Store the chromatin at -80°C for up to 2 months for further use in the immunoprecipitation.

5. Magnetic Immunoprecipitation

Note: This protocol has been optimized for 1 million cells per ChIP. Although it is possible to use more cells, we

recommend performing separate ChIP reactions and pool the samples before purification of the DNA.

Determine the total number of IPs in the experiment. We recommend to include one negative control in each

experiment (IP with the IgG negative control). Take the required amount of protein A-coated magnetic beads (20

µL/IP).

Dilute the 5X ChIP buffer B with ChIP-seq grade water to obtain 1X ChIP buffer B. The total amount of 1X ChIP buffer

B needed is 9X the volume of beads required for the experiment. Place the diluted ChIP buffer on ice.


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Wash the beads 4X with 2X volume of ice-cold 1X ChIP buffer B. To wash the beads, add 1X ChIP buffer, resuspend

the beads by pipetting up and down several times and place the tubes in a suitable magnetic rack for 1.5-mL tubes.

Wait for one min to allow the beads to be captured by the magnet and remove the supernatant.

Repeat the above 3X. Alternatively, centrifuge the tubes for 5 min at 1,300 rpm, discard the supernatant and keep

the bead pellet.

After the last wash, resuspend the beads in the original volume of 1X ChIP Buffer.

Prepare the ChIP reaction mix according to the table below.

No. of IPs

5% BSA

200X Protease Inhibitor Cocktail

5X ChIP Buffer B

Sheared Chromatin (1x1010 cells)

Magnetic Beads

ChIP-seq Grade Water

Antibody

1 6 µL 1.5 µL 56 µL 100 µL 20 µL (116.5 – V) µL V µL





Note: If required, sodium butyrate NaBu, a potent deacetylase inhibitor, (20 mM final concentration) or other inhibitors

can also be added.

Add the antibody to the reaction mix. Use 2 µL of the rabbit IgG control antibody for the negative control IP. If a

positive control IP is included in the experiment, use 1 µL of the H3K4me3 ChIP-grade control antibody.

When preparing the reaction mix, place 1 µL of the sheared chromatin aside to be used as an input the next day.

Incubate the tubes overnight at 4°C under constant rotation at 40 rpm.

The next morning, after the overnight incubation, briefly spin the tubes and place them in a magnetic rack. Wait for

one min and remove the supernatant.

Wash the beads by adding 350 µL of wash buffer A, gently shaking the tubes to resuspend the beads, and incubating

for 5 min at 4°C under constant rotation at 40 rpm.

Repeat the wash as described above 1X with wash buffers B, C and D, respectively.

6. Elution, Decross-Linking and DNA Isolation

After removing the last wash buffer, add 400 µL of elution buffer A to the beads and incubate for 30 min under constant

rotation at room temperature.

Briefly spin the tubes and place them in a magnetic rack.

Transfer the supernatant to a new tube and add 16 µL of elution buffer B. Also add 399 µL elution buffer A and 16 µL

of elution buffer B to the 1 µL input sample kept aside the day before.

Incubate for 4 hours in a thermomixer at 1,300 rpm and 65°C. If required, the incubation at 65°C can be performed

overnight.

Pool samples if necessary.

Note: Up to two samples can be easily pooled. If more than two samples need to be pooled, process each sample

purification individually, pool final eluates at the end of the magnetic bead purification and concentrate.

Add 2 µL of carrier to each IP and input sample.


Add 400 µL of 100% isopropanol to each IP and input sample.


Note: Following the addition of isopropanol the solution may become cloudy. This is not detrimental to your experiment

and will not influence the quality or quantity of your purified DNA.

Resuspend the high-DNA recovery magnetic beads and transfer 15 µL to each IP and input sample (Final volume

should be 817 µL per reaction).


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Note: Keep the beads in liquid suspension during storage at 4°C and at all handling steps, as drying will result in

reduced performance.

Incubate IP and input samples for 1 hour at room temperature on a rotating wheel (40 rpm).

For 24 reactions, prepare the wash buffer 1 containing 50% isopropanol by mixing 2 mL wash buffer 1 w/o isopropanol

and 2 mL isopropanol (100%). Never leave the bottle open to avoid evaporation. For 100 reactions, mix 8 mL wash

buffer 1 w/o isopropanol with 8 mL isopropanol (100%).

Briefly spin the tubes, place them in a suitable magnetic rack for 1.5-mL tubes, wait 1 min and discard the buffer.

Add 100 µL wash buffer 1 per tube. Close the tubes, invert the 8-tube strip to resuspend the beads and incubate for

5 min at room temperature on a rotating wheel (40 rpm).



For 24 reactions, prepare the wash buffer 2 containing 50% isopropanol as follows by mixing 2 mL wash buffer 2 w/o

isopropanol and 2 mL isopropanol (100%). Never leave the bottle open to avoid evaporation. For 100 reactions, mix

8 mL wash buffer 2 w/o isopropanol with 8 mL isopropanol (100%).

Briefly spin the tubes, place in a suitable magnetic rack for 1.5-mL tubes, wait 1 min and discard the buffer.

Add 100 µL wash buffer 2 per tube. Close the tubes, invert the 8-tube strip to resuspend the beads and incubate for

5 min at room temperature on a rotating wheel (40 rpm).



Briefly spin the tubes and place them a suitable magnetic rack for 1.5-mL tubes, wait 1 min and discard the buffer.

Keep the captured beads and add 25 µL of elution buffer C. Close the tubes, invert the 8-tube strip to resuspend the

beads and incubate for 15 min at room temperature on a rotating wheel (40 rpm).

Resuspend the pelleted beads using the pipet and make sure that you drop them on the bottom of the tube.

Spin the tubes and place them into a suitable magnetic rack for 1.5-mL tubes, wait 1 min and transfer the supernatants

into a new labelled 1.5 mL tube. Keep the bead pellets on ice.

Repeat the elution of the bead pellets for 15 min at room temperature on a rotating wheel (40 rpm) in 25 µL elution

buffer C.

Spin the tubes and place them into a suitable magnetic rack for 1.5-mL tubes, wait 1 min and pool the supernatant

with the corresponding IP or input sample (1.5 mL tube). Discard the beads.

Note: Total elution volume for both IP and input samples is 50 µL (1.5 mL tube).

Place the DNA on ice and proceed to any desired downstream applications or store it at -20°C or -80°C until further

use.

Take 5 µL (10%) of immunoprecipitated DNA and determine the concentration with, for example, the Quant-iT High

Sensitivity dsDNA Assay Kit and Qubit fluorometer.

Store the DNA at -20°C until you are ready to analyze it with qPCR or by high throughput sequencing.

Note: The elution buffer C is suitable for direct qPCR analysis, whole genome amplification, chip hybridization and

Next-Generation sequencing.

7. Quantitative PCR Analysis

Before sequencing the samples, we recommend analyzing the immunopreciptated DNA by qPCR using at least one

positive and one negative control target. The kit contains a positive (GAPDH SS) and negative (Myoglobin exon 2) control

primer pair which can be used for the positive control antibody provided in the kit (ChIP-seq grade H3K4me3 antibody) in

SYBR Green qPCR assay using the protocol described below. Use your own method of choice for analyzing the

appropriate control targets for your antibodies of interest.

Prepare the qPCR mix as follows (20 µL reaction volume using the provided control primer pairs):


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10 µL of a 2X SYBR Green qPCR master mix

1 µL of primer mix

4 µL of water

5 µL immmunoprecitated or input DNA

Carry out the PCR with this sequence:

Denaturation step: 95°C (3 to 10 min)

40 cycles of 95°C (30 sec), 60°C (30 sec) and 72°C (30 sec).

Note: Check carefully supplier’s recommendations about Taq polymerase activation time. These PCR conditions may

require optimization depending on the type of Master Mix or qPCR system used.

8. ChIP-seq

This protocol has been optimized for ChIP-seq on an Illumina GAIIx and Ion Torrent next-generation sequencers.

However, other sequencing systems such as the Illumina HiSeq or the Thermo Fisher SOLiD systems can also be used.

Refer to their sequencing protocols for the generation of sequencing data.

chip-seq kit for histones (high sensitivity) · 2017. 11. 18. · 9. sonicator tubes 10. gloves 11....

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