blueprint antibodies validation sop · 2014. 11. 16. · do280 nm - jan 2, 2007- geraldine goens -...

BLUEPRINT ANTIBODIES VALIDATION SOP

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BLUEPRINT ANTIBODIES VALIDATION

STANDARD OPERATING PROCEDURE

Date of emission : May 16, 2011

Date of revision : November 2, 2012

R&D EPIGENETICS / ANTIBODIES PRODUCTION UNIT

Written by : Jan Hendrickx – Antibody QC Coordinator & Mustafa Tammoh

Approved by : Dominique Poncelet – R&D Epigenetics Manager


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I) PURPOSE OF THE SOP

The purpose of this SOP is to allow the production, the selection and the validation of ChIP-seq grade antibodies (polyclonal or monoclonal) for the BLUEPRINT Consortium in a standardized way.

II) WORKFLOW

For each of the targets, double branched as well as simple peptides were designed and ordered from two different suppliers and up to 30 rabbits were immunized. The immune response and specificity of all bleeds was tested by ELISA, dot blot, WB and ChIP. Only those bleeds that passed this initial QC were purified. The purified antibodies were tested again for immune response and specificity. Purified antibodies that passed this QC and gave similar results in ChIP were pooled and tested in ELISA, dot blot, peptide array, WB, IF, ChIP and ChIP-seq. In general, only purified antibodies originating from the same rabbit were pooled. These pools constitute the final BP lots. This SOP describes the characterization of the final lots.


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III) QUALITY CONTROL PROCESS

1) ELISA

ELISA is performed to determine the titer of the antibody. A 3-fold dilution series of the antibodies ranging from 1/50 to 1/36450 is tested. The ELISA plates are coated with the peptide that was used to generate the antibody. Only antibodies with a titer >3000 are retained for the BP project. A detailed ELISA protocol can be found below.

Related Documents: - W:\@Protocols\SOP Word version\ SOP-RED-001 ELISA Assay Protocol V001EN.doc - W:\@Protocols\SOP Word version\ ANNEX 2 - ELISA Criteria V001.doc - W:\@Protocols\SOP Word version\SOP ARCHIVE\SOP RED 009 Determ conc Pab par DO280 nm - Jan 2, 2007- Geraldine Goens - supplier datasheets Material & reagents:

- 96 wells High Binding microplate (Greiner BioOne, Ref: 655.081) - Coating buffer (Sigma, Ref: C3041-50CAP). Dissolve 1 capsule in 50 ml to obtain a

Carbonate-bicarbonate buffer 0.1M pH 9,6 - TRIS-HCl 1M pH8 (GIBCO Invitrogen, Ref: 15568-025) - DMSO 10% (Sigma, Ref: 276855-100M) - KLH 10 mg/ml (Sigma, Ref: H7017). - PBS-Tween (PBST): 0,001M KH2PO4 / 0,0027M KCl / 0,01M Na2HPO4 / 0,14M NaCl +

0.05 % v/v ProClin 300 (Sigma, Ref: 48912-U) + 0.05 % v/v Tween 20 (Sigma, Ref: P5927-100ML) pH 7.4

- BSA (Sigma, Ref: 05477) - Saturation Solution: 3% BSA-PBST (1.5 g BSA in 50 ml PBST) - Dilution Buffer : 1% BSA-PBST (15 ml of 3% BSA-PBST in 30 ml of PBST) - ELISA Wash Solution: PBST - Goat Anti Rabbit HRP conjugated (Sigma, Ref: A9169) diluted 1:100 in Guardian (Pierce,

Ref: 37548) (storage max 1 year after preparation at 4°C) - TMB (Sigma, Ref: T0440)


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- Stop Solution: Sulfuric acid 25 % ~3M (Rectapur, Ref: 84.513.290) dilute 3 times with deionised water to obtain a 1M solution

- ELISA Plate reader (Bio-Rad Microplate Reader, model 680 # 168-1000)

Protocol:

- Dilute the peptide in TRIS-HCl 50mM pH8 filtered on 0.22 µ to obtain a concentration of 10 mg/ml. Dilute hydrophobic peptides to a concentration of 1mg/ml in TRIS-HCl 50mM pH8 containing 10% DMSO.

- Add 100 µl / well (100 ng/well) of coating solution (peptide, KLH, IgG Rabbit) Peptide: 100 µl / well (Strips 1-2, 5-6 & 8-9) KLH: 100 µl / well (Strips 3-4, 7-8 & 11) Positive Control: 100 µl / well (Strip 12, Wells E, F, G & H) Negative Control: empty wells (Strip 12, Wells A, B, C & D)

- Cover with a Micro-titer Sealing Tape and Incubate overnight at 4°C - Wash twice with ELISA Wash Solution and dry on paper - Add 110 µl Saturation Solution / well and incubate 2 hours ± 15 min at room temperature - Wash once with ELISA Wash Solution and dry on paper - Add 20 µl antibody to 980 µl dilution buffer (dilution 1/50); add 42 µl of the Flowthrough to

958 µl dilution buffer. - Add 150 µl of the samples 1/50 dilution in duplicate to the plate

Crude serum: 150 µl / well (strips 1-4, wells A) Purified antibody: 150 µl / well (strips 5-8, wells A) Flowthrough: 150 µl / well (strips 9-11, wells A)

- Add 100 µl dilution buffer in all the wells except in wells A, strips 1-11 - Using a multichannel pipet, perform a serial dilution in Strips 1-11 by taking 50 µl from

the wells A and transferring it to wells B. Then take 50 µl from the wells B and transfer it to wells C and so on. Mix well when transferring from well to well and throw away the last 50 µl after the last mix in wells G wells A: dilution 1/50 wells B: dilution 1/150 wells C: dilution 1/450 wells D: dilution 1/1,350 wells E: dilution 1/4,050 wells F: dilution 1/12,150 wells G: dilution 1/36,450 wells H: contains only dilution buffer and is used to determine the level of non- specific binding


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- Cover with a Micro-titer Sealing Tape and incubate overnight at 4°C - Wash 7 times with deionised water and dry on paper - Add 100 µl / well of Goat Anti-Rabbit conjugated HRP at final dilution of 1: 100,000

(Dilute 1:1,000 of conjugate HRP already diluted 1:100 in Guardian) - Cover with a Micro-titer Sealing Tape and Incubate 90 minutes ± 15 minutes at room

temperature - Wash 7 times with deionised water and dry on paper - Add 100 µl / well of TMB - Incubate 30 minutes ± 5 minutes at room temperature. Switch on the Microplate Reader

(has to be done at least 15 minutes before use) - Add 100 µl / well of Stop Solution - Read immediately at 450 nm on a ELISA Plate reader - Export results (Raw Data) on “REC RED 012 QC Elisa Purif Results Matrix.xls” and save

with the appropriate name (target and lot#)

2) DOT BLOT The specificity of antibodies against modified proteins (e.g. histones) is tested by dot blot. For dot blot, 8 to12 relevant peptides are spotted on the blot in different amounts ranging from 100 to 0.2 pmol. These peptides include other modifications of the same residue, similar modifications of other residues, and the unmodified sequence. The antibody is tested at a dilution of 1/20 000. Depending on the result the concentration can be increased or decreased (1/1 000 - 1/200 000). The criterium applied for the BP antibodies is that the signal obtained with the specific peptide should be >90% of the total signal on the blot for the highest peptide concentration. A detailed protocol for dot blot is described below. Related Documents: - W:\@Protocols\SOP Word version\SOP-RED-005 Dot blot Protocol - V001.doc

Material & reagents:

- 96 wells plate, none or low binding (NUNC, Ref: 269620) - PVDF membrane (GE Healthcare, Ref: RPN303F) - Methanol 99,9% (Sigma, Ref: 34860-2,5l-R) - Deionised water (VWR Prolabo, Ref: #23597.410)


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- Tris Buffered Saline 10X (TBS 10X) - Tween 20 (Sigma, Ref: P5927) - Non Fat Milk powder (Régilait, Supermarket) - TBS-Tween 0.05 % (TBS-T): mix 100 ml TBS 10X with 900 ml deionised water and add

500 µl Tween 20 - TBS-T 5% Milk: Add 5 g of non fat milk powder to 100 ml of TBS-T - Stock peptide at 5 mM or another concentration - Primary antibody diluted into TBS-T 5% non Fat Milk

(working dilution depends of antibody titre, 1:20,000 could be the starting dilution but an evaluation should be done depending on ELISA results)

- Secondary antibody: ECL Peroxidase labelled anti-rabbit (GE Healthcare, Ref: NA934VS)

- Substrate: ECL Advance Western Blotting Detection Kit (GE Healthcare, RPN2135) - KODAK Imaging system GEL LOGIC 1500 for fluorescence

Protocol:

- Add 990 µl of Tris 50 mM in 10 µl of 5 mM stock peptide to obtain a peptide concentration of 50 pmol/µl

note: this solution of peptides can be aliquoted and kept at -20°C - In a 96 wells plate, add Tris 50 mM in the different wells as describes in the next figure

(one row per peptide)

A

B

C

D

E

F

G

H

100 µl

100 µl

240 µl

240 µl

240 µl

100 µl

- Add 200 µl of each diluted peptide in the wells A - Make a serial dilution as describes in the figure below:


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100 µl

60 µl

60 µl

60 µl

100 µlA

B

C

D

E

F

G

H note: Several membranes can be spotted and stored (dried between two filter papers) during several weeks (one aliquot of 10 µl of Stock peptide is enough for about 200 membranes)

- Cut the PVDF membrane (size: 11,8 cm/ 7 cm) - Wet a filter paper with TBS 1X - Re-hydrate the membrane 1 minute in methanol 100 % - Wash the membrane 5 minutes in deionised or distilled water - Wash the membrane 10 seconds in TBS 1X - Place the wet filter paper on a plane surface - Place membrane on the filter paper - Spot each dilution of peptide on the membrane (drops of 2 µl)

- Re-hydrate the membrane 1 minute in methanol 100 % - Transfer the membrane in a common TBS-T 5% Non Fat Milk to remove the methanol - Wash the membrane 1 hour at room temperature and under agitation with TBS-T 5%

Non Fat Milk (Blocking non specific binding) - Incubate the membrane 1 hour at room temperature and under agitation with primary

antibody diluted 1/20.000 (or another dilution) into TBS-T 5% Non Fat Milk


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- Wash 3 times the membrane 10 minutes under agitation with TBS-T 5% Non Fat Milk - Incubate the membrane 1 hour at room temperature under agitation with the secondary

antibody diluted 1/20.000 with TBS-T 5% Non Fat Milk - Wash 3 times the membrane 10 minutes under agitation with TBS-T - Reveal the membrane with the ECL Advance Western blotting detection Kit. (Mix 1

volume of solution A with 1 volume of solution B, 750 µl of mix is necessary per membrane)

- Apply 750 µl of mix all over the membrane and take a picture with the KODAK Imaging system

3) MODIFIED HISTONES PEPTIDE ARRAY

The specificity of antibodies against modified histones is further tested on Active Motif peptide arrays. These arrays contain 384 different peptides in duplicate with different combinations of H3, H4, H2A and H2B modifications. The antibodies are used at a dilution from 1/2000 to 1/10000. After performing the array, the specificity factor, the ratio of the average intensity of all spots containing the mark, divided by the average intensity of all spots not containing the mark is calculated. A specificity factor >30 and at least 5x higher than for any other modification is a required for the BP antibodies. A detailed peptide array protocol is described below. Related documents: - Active motif’s instruction manual: Modified Array Labeling Kit (Ref: #13006) - W:\@Protocols\SOP Word version\ SOP-RED-017 Peptide Array Protocol - V001.doc Material & reagents:

- Modified Histone Peptide Arrays (Active Motif, Ref: #13005) - Blocking Buffer - 10X Wash buffer - c-Myc mouse monoclonal antibody (positive control) - Anti-mouse HRP-conjugated secondary antibody - Anti-rabbit HRP-conjugated secondary antibody - ECL Reagent A - ECL Reagent B


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- Primary antibody - NUNC Well Rectangular Dishes (NUNC, Ref: 267061) or suitable chamber for array

incubation - KODAK Imaging system GEL LOGIC 1500 for fluorescence - Active’s motif’s Array Analyze Software program (available for download at

www.activemotif.com/modified) Protocol:

- Thaw the blocking buffer prior to starting the assay. Once thawed, the buffer should be stored at 4°C and used within 24 hours. Otherwise, the buffer should be frozen at -20°C.

- Immerse one array in 3 ml blocking buffer Note: if working with multiple arrays at the same time, “4 well rectangular dishes” from NUNC can be used. The dish does not contain a lid, so it is recommended to use a low setting on an orbital shaker to prevent contaminations.

- Incubate on an orbital shaker for 3 hours at room temperature. - Meanwhile, prepare the amount of 1X Wash buffer required for the labeling assay as

follows: for every 100 ml of 1X Wash buffer, dilute 10 ml 10X Wash buffer with 90 ml distilled water. Mix gently to avoid foaming. The 1X Wash buffer may be stored at 4°C for one week. The Tween 20 contained in the 10X buffer may form clumps. Therefore it is necessary to completely resuspend any precipitates by incubating at 50°C for 2 minutes and mixing prior to use.

- Carefully pour off the buffer, perform a quick rinse with 5 ml 1X wash buffer. Then wash three times for 5 minutes on the orbital shaker using 5 ml 1X wash buffer

- During the last wash, dilute the primary antibody combined with the c-Myc antibody in 3 ml blocking buffer. Tested antibodies should be diluted at a 1:2,000 to 1:10,000 dilution. The control c-Myc antibody is provided for the detection of c-Myc tag at location P21 on the array. The c-Myc antibody can be combined directly with the sample antibody at a 1:2,000 dilution

- Add the antibody solution to the array and incubate for 1 hour at room temperature. Set the orbital shaker to a low setting to prevent any cross contamination.

- Carefully pour off the antibodies solution and perform a quick rinse with 5 ml 1X wash buffer. Then wash three times for 5 minutes on the orbital shaker using 5 ml 1X wash buffer.

- During the last wash, prepare the HRP-conjugated secondary antibodies: Both the anti-rabbit and anti-mouse HRP-conjugated antibodies should be diluted 1:2,500 in 3 ml blocking buffer.


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- Add the conjugated secondary antibody solution to the array and incubate for 1 hour at room temperature. Set the orbital shaker to a low setting to prevent any cross contamination.

- Perform a quick rinse with 5 ml 1X wash buffer. Then wash three times for 5 minutes on the orbital shaker using 5 ml 1X wash buffer.

- During the last wash, prepare the detecting solution by diluting 1,5 µl of ECL reagent A in 5 ml of ECL reagent B. The solution should be prepared just before use and kept protected from light.

- The array is incubated in the detecting solution for 5 minute at room temperature. - Use a CCD camera to capture images at multiple exposure times (10, 30 & 60 sec.). It’s

also recommended to take a white light image of the array in order to obtain orientation information for the analysis step.

- Save the image file and perform the array analysis using the “Active Motif’s software”.

4) WESTERN BLOT

The overall specificity of the antibodies is tested in Western blot. Western blot was performed on whole cell extracts and histone extracts from HeLa cells and on 4 recombinant histones H2A, H2B, H3 and H4. The antibody is initially tested at a dilution of 1/1000. Depending on the result the concentration can be increased or decreased (1/200 - 1/4000). The following criteria are applied for BP antibodies: - a specific signal >80% of the total signal in the lane containing the whole cell extracts - signal of other histones


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- Migration buffer Tris/Glycine/SDS 10X (Biorad, Ref: 161-0732) - Laemmli Sample Buffer (Biorad, Ref: 161-0737) - Β-mercaptoethanol (Biorad, Ref: 161-0710) - Recombinant protein MW marker “Full range rainbow” (GE Healthcare, Ref: RPN800E) - PVDF membrane (GE Healthcare, Ref: RPN303F) - Methanol 99,9% (Sigma, Ref: 34860-2,5l-R) - Transfer Buffer Tris/Glycine 10X (Biorad, Ref: 161-0734) - Deionised water (VWR Prolabo, Ref: #23597.410) - 10X Tris Buffered Saline (TBS 10X)(Biorad, Ref: 170-6435) - Tween 20 (Sigma, Ref: P5927) - Non Fat Milk powder (Régilait, Supermarket) - TBS-Tween 0.05 % (TBS-T): mix 100 ml TBS 10X with 900 ml deionised water and add

500 µl Tween 20 - TBS-T 5% Milk: Add 5 g of non fat milk powder to 100 ml of TBS-T - Primary antibody diluted into TBS-T 5% Non Fat Milk

(working dilution depends of antibody titre, 1:1.000 could be the starting dilution but an evaluation should be done depending on ELISA results)

- Secondary antibody: ECL Peroxidase labelled anti-rabbit (GE Healthcare, Ref: NA934VS)

- Substrate: ECL Advance Western Blotting Detection Kit (GE Healthcare, RPN2135) - Mini Protean 3 Electrophoresis Module & Mini Trans-blot Electrophoretic Transfer Cell

(Biorad) - KODAK Imaging system GEL LOGIC 1500 for fluorescence

Protocol:

- Remove carefully the comb of the gel - Rinse the wells with deionised water - Place the glass plates containing the gels in the Biorad migration cassette - Fill the inner reservoir with 125 ml and the outer reservoir with 200 ml of Running Buffer

1X - Bring water to boil - Prepare Laemmli Sample Buffer containing 5 % of Β-mercaptoethanol - For the histone marks take 25 µg whole cell extracts, 15 µg histone extracts & 1 µg

recombinants histones (H2A, H2B, H3.1 & H4). Add an equal volume of Laemmli buffer containing B-mercaptoethanol. For Nuclear proteins take 40 µg of nuclear extracts & 25 µg whole cell extracts and add an equal volume of Laemmli buffer containing B-mercaptoethanol. PS: the protein standard is ready to use (5 µl per lane and per gel)

- Boil the samples (included the protein standard) for 2 minutes


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- Load the wells - Migrate at 200V for 35 minutes or until the blue band reaches the bottom of the gel. - Soak the filter paper and the fiber pads in transfer buffer. - Cut the PVDF membrane (8,5 x 6 cm) - 5 minutes before the end of the migration, rehydrate the membrane for 1 minute in

methanol and equilibrate for 10 minutes in transfer buffer - After migration, the gel is equilibrated in transfer buffer during minimum 5 minutes - The gel and the membrane (on top of the gel) is placed in the sandwich:

Fiber pad - filter paper – gel – membrane – filter paper – fiber pad - Put everything in the transfer system and mind the correct orientation of the

anode/cathode. Don’t forget the ice block - Start the transfer at 100V during 1 hour - The membrane is blocked with TBS-T containing 5% milk powder during 1h at room

temperature - The primary antibody is diluted in TBS-T 5 % milk powder at a 1/1.000 dilution (or

another dilution in function of the titre) - Incubation over night at 4°C - Wash the membrane 3X10 minutes in TBS-T + 5% milk powder - The secondary antibody is diluted in TBS-T 5% milk powder at dilution 1/10.000 - The membrane is incubated for 1h in the TBS-T containing 5 % milk powder containing

the secondary antibody - The membrane is washed 3X10 minutes in TBS-T - The solution for revelation is prepared (ECL advance Western Blot detection Kit: 750µl

solution A + 750 µl solution B = 1,5 ml for two membranes) - Incubate the membrane with the solution for revelation for 30 seconds - Make a picture with the KODAK system with the following parameters

exposure time: 30 seconds illumination source: luminescence f-stop : 2,8 zoom : 30 Excitation : none Filter : none

5) CHROMATINE IMMUNO-PRECIPITATION ASSAY

ChIP is performed on sheared chromatin from 106 HeLa cells with the Auto Histone ChIP-seq kit. The separate purified antibodies are tested in two different amounts (1 and 5 µg per IP); the pooled purifications are tested in 1, 2, 5 and 10 µg per IP. If necessary, lower amounts of


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antibody are tested also. qPCR is performed using at least 2 positive and 2 negative control targets. To pass the ChIP QC, the antibody has to show the expected profile with a +/- ratio > 5. The recovery of the positive control targets should be > 1%. The protocol below describes the manual ChIP with the Auto Histone ChIP-seq kit. Alternatively, the IP-Star Compact automated system can be used.

Related documents: - manual Auto Histone ChIP-seq Kit (Diagenode, Ref: AB-auto02-A100) - W:\@Protocols\SOP Word version\SOP-RED-016 ChIP Protocol - V001.doc Material & Reagents

- DiaMag1.5 magnetic rack (Diagenode, Ref: kch-816-015) - Rotating wheel - Thermomixer - Protein A coated magnetic beads - ChIP buffer H - Rabbit IgG - Protease inhibitor mix 200X - Wash buffers H1, H2, H3 & H4 - Elution buffer H - NaCl 5M - IPure Kit (Diagenode, Ref: AL-100-0100)

Protocol

1) cell fixation and chromatin shearing according to the protocol from the HighCell# ChIP kit to obtain 107 cells in 200 µl of shearing buffer S1

2) ChIP according to the Auto Histone ChIP-seq kit

manual protocol: - take the required amount of magnetic beads (20 µl/IP) and wash 4 times with 1 ml of ChIP buffer H using the magnetic rack - dispense the magnetic beads in n x 100 µl (with n = number of IP’s) and divide into the required number of tubes (100 µl/tube)


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- add the antibody (1 & 5 µg for each of the 4 separate purifications and 1, 2, 5 & 10 µg for the pool of purifications). - incubate at 4°C on a rotating wheel for 4 hours. - dilute the chromatin 1/10 in ChIP buffer H + protease inhibitor 200X to obtain a total volume of 200 µl per IP; take an excess of 100 µl to be used as input (e.g. for 8 IP’s use 170 µl chromatin, 8.5 µl Protease inhibitor and 1521.5 µl buffer H) - remove the buffer from the beads using the magnetic rack and add 200 µl of the diluted chromatin - incubate overnight at 4°C on a rotating wheel - wash the beads for 5’ at 4°C once with 150 µl of each of the washing buffers H1, H2, H3 and H4 - remove the washing buffer after the last wash and add 100 µl of elution buffer to the beads; add 98 µl of elution buffer to 2 µl of input - incubate for 20 - 30’ at room temperature on a rotating wheel - take the elution buffer from the beads and transfer it to a new tube; add 4 µl of 5M NaCl; also add 4 µl of 5M NaCl to the input - incubate for 4 hours at 65°C and 1300 rpm in a thermomixer - Purify the recovered DNA using the Diagenode’s IPure Kit - Perform a quantitative PCR and analyse the results automated protocol: - switch on the IP-Star and select “ChIP protocol - Direct method - for 1-8 samples select ChIP_8_IPure_200-D, for 9-16 samples select ChIP_8_IPure_200-D - choose the nr. of samples - select 4 hours for antibody coating, 15 hours for IP reaction and 5’ for washing - follow the instructions on the screen - when the IP is finished remove the strip containing the samples (row 12) - take the elution buffer from the beads and proceed as described in the manual protocol

6) ChIP-SEQUENCING


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ChIP for ChIP-seq is performed on sheared chromatin from 105 K562 cells with the iDeal ChIP-seq kit. The antibodies are tested in different amounts of 0.2, 0.5, 1 and 2 µg per IP. IgG (1 µg/IP is used as a negative control. qPCR is performed using at least 2 positive and 2 negative control targets. The optimal antibody concentration is determined based on the recovery, the +/- ratio and the amount of precipitated DNA. If necessary, different ChIP’s on 100 000 cells are pooled to obtain sufficient material for sequencing. The ChIP’d DNA is sequenced on an Illumina GAIIex. Library preparation, cluster generation and sequencing are performed using the standard Illumina protocols. The sequences are aligned to the human genome (hg18 or hg19 depending on the antibody) using ELAND or BWA. For data analysis the results are uploaded in the UCSC genome browser and compared to publicly available data from the Broad Institute. For this comparison, a bioinformatic analysis is performed. This bioinformatic analysis includes peak detection on the Blueprint antibodies and on the Broad data using SICER with the same settings. These settings are optimised for each mark individually. The % of reads in peaks (RIP) is calculated and has to be at least 40% and at least as high as the % RIP obtained with the corresponding Broad data. Further, the peaks obtained with the Blueprint antibodies and with the Broad data are compared, both the complete set of peaks in the genome and the top 40% most significant peaks. This overlap has to be at least 70% for the complete set of peaks, and at least 90% for the top 40 peaks.

Related documents:

- manual iDeal ChIP-seq Kit (Diagenode, Ref: AB-001-0024) - Preparing Samples for ChIP Sequencing of DNA - TruSeq Cluster Generation Kit v5 Reagent Preparation Guide For Single-Read Runs (Illumina document) - Single-Read Sequencing on the Genome Analyzer experienced user card (Illumina document) - manual QIAquick PCR purification kit (Qiagen, 28106) - W:\@Protocols\SOP Word version\Illumina library preparation - manual TruSeq Cluster Generation Kit v5 (Illumina, GD-203-5001) - manual TruSeq ChIP Sample Preparation Kit - Set A (Illumina, IP-202-1012) - manual NEXTFlex ChIP-seq kit (BIOO Scientific, 5143-02) A. ChIP

Materials & Reagents


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- iDeal ChIP-seq Kit (Diagenode, Ref: AB-001-0024) - DiaMag1.5 magnetic rack (Diagenode, Ref: kch-816-015) - Rotating wheel - Thermomixer - QIAquick PCR purification kit (Qiagen, cat. No. 28106)

STEP 1. Cell collection and DNA-protein crosslinkin g 1. Collect the cells by trypsinisation and wash two times with PBS. 2. Count the cells and resuspend them in PBS to obtain 10 million cells in 500 µl of PBS. Aliquot 500 µl of cell suspension in 1.5 ml tubes. 3. Add 13.5 µl of formaldehyde. Mix by gentle vortexing and incubate for 8 minutes at room temperature to allow fixation to take place. 4. Stop the fixation by adding 57 µl of Glycine solution. Mix by gentle vortexing and incubate for 5 minutes at room temperature. Work on ice from this point onwards. 5. Centrifuge at 1,600 rpm (250 x g) for 5 minutes at 4°C and gently aspirate the supernatant without disturbing the cell pellet. 6. Wash the cells twice with 1 ml PBS. STEP 2. Cell lysis and chromatin shearing 7. Add 10 ml of ice-cold Lysis Buffer L1 to the cell pellet. Resuspend the cells by pipetting up and down several times and incubate for 10 minutes at 4°C with gentle mixing. 8. Centrifuge for 5 minutes at 1,600 rpm (500 x g) and 4°C and discard the supernatant. 9. Add 10 ml of ice-cold Lysis Buffer L2 to the cell pellet. Resuspend the cells by pipetting up and down several times and incubate for 10 minutes at 4°C with gentle mixing. 10. Centrifuge for 5 minutes at 1,600 rpm (500 x g) and 4°C and discard the supernatant. 11. Add 200x protease inhibitor mix to the Shearing Buffer S1. Keep the buffer at room temperature until use. 12. Add 1 ml of Shearing Buffer S1 containing protease inhibitor to the cells. Resuspend by pipetting up and down. 13. Shear the chromatin by sonication using the Bioruptor® for two runs of 10 cycles [30 seconds “ON”, 30 seconds “OFF”] each. Briefly vortex and spin the tubes between the runs. 14. Centrifuge at 13,000 rpm (16,000 x g) for 10 minutes and collect the supernatant which contains the sheared chromatin. STEP 3. Magnetic Immunoprecipitation 15. Dilute 1 ml of 5x ChIP buffer C1 and with 4 ml MilliQ water to obtain 1x ChIP buffer. Place on ice.


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16. Take the required amount of ProtA coated magnetic beads (20 µl/IP) and wash them four times with 1 ml of ice-cold 1x ChIP buffer C1. 17. Resuspend the beads after the last wash in the original volume of 1x ChIP Buffer C1. 18. Label the required number of tubes and pipet 20 µl of the resuspended beads in each tube. 19. Prepare the following ChIP reaction mix: 6 x n µl 5% BSA, 1.5 x n µl 200x PI, 56 x n µl 5x ChIP buffer C1, 10 x n µl sheared chromatin, 90 x n µl shearing buffer, 115.5 x n µl water, with n = the number of samples +1. 20. Add 279 µl of the ChIP reaction mix to the tubes containing the beads. Keep the remaining reaction mix to be used as an input the next day. 21. Add the required amount of antibody (~1 µl) 22. Incubate overnight at 4°C on a rotating wheel. 23. Briefly spin the tubes, place them in the ice-cold magnetic rack and discard the supernatant. 24. Add 350 µl ice-cold Wash buffer W1 and incubate for 5’ at 4°C on a rotating wheel. Discard the wash buffer using the magnetic rack. 25. Repeat step 20 and 21 once with Wash buffer W2, W3 and W4, respectively. STEP 4. Elution, decrosslinking and DNA isolation 26. After removing the last wash buffer, add 400 µl of elution buffer E1 to the beads and incubate for 30’ on a rotating wheel at room temperature. 27. Briefly spin the tubes and place them in the Magnetic Rack. Transfer the supernatant to a new tube and add 16 µl of NaCl. Also add 397 µl buffer E1 and 16 µl NaCl to 2.8 µl of the input sample. Incubate for 4 hours in a thermomixer at 1300 rpm and 65°C. 28. Purify the DNA with the Qiaquick PCR purification kit. If necessary, pool different samples and purify them on the same column. 29. Analyze the samples by QPCR and measure the concentration using the Qubit DNA HS assay kit. B. library preparation

1. Perform End Repair Consumables ChIP enriched, qPCR verified DNA (5-20 ng) T4 DNA ligase buffer with 10 mM ATP (Promega, cat. No. C126A) 10 mM dNTPs mix (composed of Roche cat. No. 11934511001, 11934520001, 11934538001 and 11934546001) T4 DNA polymerase (NEB, cat. No. M0203C) Klenow DNA polymerase (NEB, cat. No. M0210) T4 PNK (NEB, cat. No. M0201)


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Water QIAquick PCR purification kit (Qiagen, cat. No. 28106)

Procedure Dilute Klenow DNA polymerase 1:5 with water. Prepare the following reaction mix: x µl ChIP enriched DNA (5-20 ng) 5 µl T4 DNA ligase buffer with 10 mM ATP 2 µl dNTP mix 1 µl T4 DNA polymerase 1 µl diluted Klenow DNA polymerase 1 µl T4 PNK Water up to a total volume of 50 µl. Incubate in the thermal cycler for 30 minutes at 20ºC. Follow the instructions in the QIAquick PCR Purification Kit to purify on one QIAquick column, eluting in 34 µl of EB. 2. Add ‘A’ Bases to the 3’ End of the DNA Fragments Consumables Klenow buffer (NEB buffer 2) 1 mM dATP (Roche cat. No. 11934511001) Klenow fragment (3’ to 5’ exo minus) (NEB, cat. No. M0212) MinElute Reaction Cleanup Kit (Qiagen, cat. No. 28204) Procedure Prepare the following reaction mix: 34 µl DNA sample 5 µl Klenow buffer 10 µl dATP 1 µl Klenow exo (3’ to 5’ exo minus) The total volume should be 50 µl. Incubate for 30 minutes at 37ºC. Follow the instructions in the MinElute Reaction Cleanup Kit to purify on one MinElute column, eluting in 11 µl of EB. 3. Ligate Adapters to DNA Fragments


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Consumables Quick ligation kit (NEB cat. No. M2200L) Adapter oligo mix (Illumina) Ultra pure water QIAquick PCR purification kit (Qiagen, cat. No. 28106)

Procedure Dilute the Adapter oligo mix 1:10 with water. Prepare the following reaction mix: 11 µl DNA sample 15 µl DNA ligase buffer 1 µl Diluted adapter oligo mix 3 µl DNA ligase The total volume should be 30 µl. Incubate for 15 minutes at room temperature. Follow the instructions in the QIAquick PCR Purification Kit to purify on one QIAquick column, eluting in 44 µl of EB. 4. Pre-Size Selection PCR. Consumables AccuPrime Pfx DNA Polymerase (Life Technologies cat No. 12344-024) 10X AccuPrime Pfx Reaction Mix PCR primer 1.1 (IDT) PCR primer 2.1 (IDT) QIAquick MinElute PCR Purification Kit (Qiagen cat. No. 28004) Procedure Prepare the following PCR reaction mix: 43.4 µl DNA 5 µl 10x AccuPrime Pfx Reaction Mix 0.6 µl PCR primer 1.1 0.6 µl PCR primer 2.1 0.4 µl AccuPrime DNA polymerase The total volume should be 50 µl. Amplify using the following PCR protocol:

2’ at 95ºC 4 cycles of: 15 seconds at 95ºC

30 seconds at 65ºC 1’ at 68ºC


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5 minutes at 68ºC Follow the instructions in the MinElute PCR Purification Kit to purify on one MinElute column, eluting in 10 µl of EB. 5. Size Select the Library This protocol removes excess adaptors and selects a size range of templates to go on the Cluster Station. Consumables 2% Agarose Gel 100 bp DNA marker (Promega) QIAquick gel extraction kit (Qiagen cat. No. 28604) Procedure Prepare a 2% agarose gel in 1xTAE. Add 2 µl gel loading dye to 10 µl sample. Load the sample in the middle of the gel; load 6 µl of 100 bp marker (Promega) in the first and the last well of the gel. Run the gel at 100V for approximately 45 minutes. Stain the gel in 1x TAE containing SYBRsafe for approximately 20 minutes. Using a UV transilluminator, cut out the fragments of ~300 bp Purify the DNA with the QIAquick gel extraction kit; elute the DNA in 44 µl. 6. Enrich the Adapter-Modified DNA Fragments by PCR Consumables AccuPrime Pfx DNA Polymerase (Life Technologies cat No. 12344-024) 10X AccuPrime Pfx Reaction Mix PCR primer 1.1 (IDT) PCR primer 2.1 (IDT) QIAquick MinElute PCR Purification Kit (Qiagen cat. No. 28004) Procedure Prepare the following PCR reaction mix: 43.4 µl DNA 5 µl 10x AccuPrime Pfx Reaction Mix 0.6 µl PCR primer 1.1 0.6 µl PCR primer 2.1


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0.4 µl AccuPrime DNA polymerase The total volume should be 50 µl. Amplify using the following PCR protocol:

2’ at 95ºC 14 cycles of: 15 seconds at 95ºC

30 seconds at 65ºC 1’ at 68ºC

5 minutes at 68ºC Follow the instructions in the MinElute PCR Purification Kit to purify on one MinElute column, eluting in 15 µl of EB. Check the library with the bioanalyzer and determine the concentration with the Qubit HS dsDNA assay kit.