Semi-Automated Chromatin Immunoprecipitation
(v1.0)
Type of Document: SOP
Document Number: ???????
Version Number: 01
Purpose of Document
Describe how to perform a semi-automated chromatin immunoprecipitation using the Diagenode IP-Star and
Diagenode Bioruptor; including relevant quality control and validation steps.
Tables of Contents
1.
1.2.
1.2.1.
1.2.2.
1.2.3.
2.
2.1.
2. .2.
3.
4.
Definitions
Consumables and Equipment
Required Equipment
Required Reagent(s)
Buffer Composition(s)
Procedure
Basic Sonication Procedure for Chromatin Immunoprecipitation
Sonication Optimization
Records
Associated documents
1. Definitions
DNA: Deoxyribonucleic Acids; SOP: Standard Operating Procedure; Min: Minute; Bp: base pair; ChIP: Chromatin
Immunoprecipitation; ddH2O: double distilled water
2. Procedure
2.1. Materials
2.1.1. Hazardous Materials
HCl: Corrosive
PMSF: Toxic
SDS: Toxic (at least to the lungs)
2.1.2. Requirements
Lab coat, protective spectacles, poweder-free gloves
2.1.3. Equipment
Description: Sonicator
Company: Diagenode
Model / Cat. #: Bioruptor UCD-300 / UCD-300 TO
Location: 6401
Description: Sonicator Water Cooler
Company: Diagenode
Model / Cat. #: Bioruptor Water Cooler / BioAcc-cool
Location: 6401
Description: SX-8G IP-Star Compact
Company: Diagenode
Model / Cat. #: SX-8G IP-Star Compact / UH-002-0001
Location: 6401
Description: Hot Block
Company:
Model / Cat. #:
Location:
Description: Nanodrop
Company:
Model / Cat. #:
Location:
Description: Q-RT-PCR
Company:
Model / Cat. #:
Location:
Description: Refrigerated Microcentrifuge
Company:
Model / Cat. #:
Location:
Description: <-80 Freezer
Company:
Model / Cat. #:
Location:
Description: <-20 Freezer
Company:
Model / Cat. #:
Location:
Description: Bioanalyzer
Company:
Model / Cat. #:
Location:
Description: <-80 Freezer
Company:
Model / Cat. #:
Location:




Pipettes (p1000, p200, p20, and p2 or equivalents)
Weight scales
pH metre
Magnetic stirrer
2.1.4. Consumables
Description: 1.5ml TPX Tubes
Company: Diagenode
Cat #: M50001
Location: 6400
Description: Non-Stick Rnase/DNase-Free 1.5ml Tubes
Company: Ambion
Cat #: AM12450
Location: 6400
Description: Non-Sterile 100-1000ul Tips
Company: Corning
Cat #: 4867
Location: 6400
Description: Non-Sterile 1-200ul Tips
Company: Corning
Cat #: 4865
Location: 6400
Description: MinElute PCR Purification Kit
Company: Qiagen
Cat #: 28006
Location: Refrigerator and 6400
Description: Agilent High Sensitivity DNA Kit
Company: Agilent
Cat #: 5067-4626
Location: Refridgerator
Description: Auto Histone ChIP-seq Kit (Protein A)
Company: Diagenode
Cat #: AB-Auto02-A100
Location: Refridgerator and <-20C Freezer
Scrapers (sterile) – quantity
Tissue Grinder (Kimble Chase) 885300-0015 – 15ml
885300-0000 – 0.5ml
885300-0001-1ml
885300-0007-7ml
2.1.5. Reagents
Name: RNase Cocktail
Description: Mixture of RNase A (500U/ml) and RNase T1 (20,000 U/ml)
Company: Invitrogen
Cat. #: AM2286
CAS #: N/A
MW: N/A
Location: Freezer 6400
Name: Proteinase K
Description: >20 units/g
Company: Invitrogen
Cat. #: 25530-015
CAS #: N/A
MW: N/A
Location: Freezer 6400
Name: Complete Mini EDTA-free Protease Inhibitors
Description: Protease inhibitor cocktail for IPB++ buffer
Company: Roche
Cat. #: 11 836 170 001
CAS #: N/A
MW: N/A
Location: 6400
Name: Phenylarsine oxide
Description: Inhibits tyrosine phosphatases
Company: Sigma
Cat. #: P3075
CAS #: 637-03-6
MW: 168.02 Da
Location: 6400
Name: Sodium orthovanadate
Description: Inhibits ATPase, alkaline phosphatase and tyrosine phosphatase
Company: Sigma
Cat. #: S6508
CAS #: 13721-39-6
MW: 183.91 Da
Location: 6400
Name: Phenylmethylsulfonyl fluoride
Description: Inhibits serine proteases
Company: Sigma
Cat. #: P7626
CAS #: 329-98-6
MW: 174.19 Da
Location: Toxic cabinet ????
Name: Sodium fluoride
Description: Inhibits protein phosphatases
Company: Sigma
Cat. #: S1504
CAS #: 7681-49-4
MW: 41.99 Da
Location: 6400
Name: IGEPAL CA-630
Description: Nonionic and non-denaturing detergent
Company: Sigma
Cat. #: I8896
CAS #: 9036-19-5
MW: N/A
Location: 6400
Name: Tris
Description: Buffer reagent
Company: Fisher
Cat. #: BP152-5
CAS #: 77-86-1
MW: 121.14 Da
Location: 6400
Name: Ethylenediaminetetraacetic acid disodium salt (EDTA) solution
Description: 0.5M solution. Metal-chelating agent; protease inhibitor.
Company: Sigma
Cat. #: E7889
CAS #: 139-33-3
MW: 336.21 Da
Location: 6400
Name: Hydrochloric Acid (HCl)
Description: 1.0N solution. Acid for changing pH of buffers.
Company: Sigma
Cat. #: H9892
CAS #: 7647-01-0
MW: 36.46
Location: Corrosive cabinet????
Name: Sodium Chloride
Description: Buffer reagent
Company: Fisher
Cat. #: S271-3
CAS #: 7647-14-5
MW: 58.44 Da
Location: 6400
Name: Sodium Dodecyl Sulfate
Description: Anionic detergent
Company: Bio-rad
Cat. #: 161-0302
CAS #: 151-21-3
MW: 288.38 Da
Location: 6400
Name: Sodium Deoxycholate
Description: Anionic detergent
Company: Sigma
Cat. #: D6750
CAS #: 302-95-4
MW: 414.55 Da
Location: 6400
Name: PIPES
Description: Buffer reagent
Company: Sigma
Cat. #: P1851
CAS #: 5625-37-6
MW: 302.37 Da
Location: 6400
Name: Glycine
Description: Crosslinking quenching reagent
Company: Sigma
Cat. #: G7126
CAS #: 56-40-6
MW: 75.07 Da
Location: 6400
Name: Sodium Hydroxide
Description: pH adjusting chemical
Company: Sigma
Cat. #: S5881
CAS #: 1310-73-2
MW: 40.00 Da
Location: 6400
Name: Phosphoric Acid
Description: pH adjusting acid
Company: Sigma
Cat. #: 438081
CAS #: 7664-38-2
MW: 98.00 Da
Location: 6400
Name: Potassium Chloride
Description: Buffer salt
Company: Sigma
Cat. #: P4504
CAS #: 7447-10-7
MW: 74.55 Da
Location: 6400
Name: Potassium Phosphate monobasic
Description: Buffer reagent
Company: Sigma
Cat. #: P5379
CAS #: 7778-77-0
MW: 136.09 Da
Location: 6400
Name: Sodium Phosphate dibasic
Description: Buffer reagent
Company: Sigma
Cat. #: S0876
CAS #: 7558-79-4
MW: 141.96 Da
Location: 6400
Name: Formaldehyde
Description: Crosslinking reagent
Company: Sigma
Cat. #:
CAS #:
MW:
Location: 6400

Liquid Nitrogen
2.2. Solutions
2.2.1. Stock Buffers
0.1 M Phenylmethylsulfonyl fluoride
1 M Phenylarsine Oxide
1 M Sodium Orthovanadate
1 M Tris-HCl, pH 8.0
5 M Sodium Chloride
1 M Sodium Floride
10% w/v Sodium Dodecyl Sulfate
10% w/v Sodium Deoxycholate
10% v/v IGEPAL CA-630
1 M PIPES, pH 8.5
10% w/v Sodium Hydroxide
1 M Potassium Chloride
1 M Glycine
10x PBS

Mix the following reagents in 800 ml ddH2O
Chemical
Sodium Chloride
Potassium Chloride
Sodium Phosphate Dibasic
Potassium Phosphate
Monobasic

Adjust pH to 7.4 using phosphoric acid

Make up to a final volume of 1 l
Amount per litre
80.0 g
2.0 g
14.4 g
2.4 g
0.5% BSA/PBS...
2.2.3. Cell Lysis Buffer



Mix the follow reagents in 2.5 ml ddH2O
Chemical
Final
Concentration
PIPES, pH 8.5 (with
5 mM
NaOH)
Potassium Chloride
85 mM
IGEPAL CA-630
1% w/v
Add the follow reagents immediately before use:
Chemical
Final
Concentration
Sodium Floride
50 mM
Phenylmethylsulfonyl
1 mM
fluoride
Phenylarsine Oxide
1 mM
Sodium Orthovanadate
5 mM
Complete Mini EDTA-free
1x tab
Protease Inhibitors
Make up to a final volume of 5 ml
Volume of Stock
per 5 ml
25 µl
Stock
Concentration
1M
425 µl
0.5 ml
1M
10% w/v
Volume of Stock
per 5 ml
250 µl
25 µl
Stock
Concentration
1M
0.2 M
20 µl
25 ul
n/a
250 mM
1M
n/a
Volume of Stock
Stock
2.2.4. Nuclei Lysis Buffer

Mix the follow reagents in 2.5 ml ddH2O
Chemical
Final


Concentration
Tris-HCl. pH 8.0
50 mM
Sodium Dodecyl Sulfate
1% w/v
EDTA
10 mM
Add the follow reagents immediately before use:
Chemical
Final
Concentration
Sodium Floride
50 mM
Phenylmethylsulfonyl
1 mM
fluoride
Phenylarsine Oxide
1 mM
Sodium Orthovanadate
5 mM
Complete Mini EDTA-free
1x tab
Protease Inhibitors
Make up to a final volume of 5ml
2.2.5. Chromatin Dilution Buffer



per 5 ml
250 µl
0.5 ml
100 µl
Concentration
1M
10% w/v
0.5 M
Volume of Stock
per 5 ml
250 µl
25 µl
Stock
Concentration
1M
0.2 M
20 µl
25 µl
n/a
250 mM
1M
n/a
(NOT NECESSARY)
Mix the follow reagents in 5ml ddH2O
Chemical
Final
Concentration
Tris-HCl. pH 8.0
50 mM
Sodium Chloride
150 mM
Sodium Deoxycholate
0.25% w/v
IGEPAL CA-630
1% w/v
Add the follow reagents immediately before use:
Chemical
Final
Concentration
Sodium Floride
50 mM
Phenylmethylsulfonyl
1 mM
fluoride
Phenylarsine Oxide
1 mM
Sodium Orthovanadate
5 mM
Complete Mini EDTA-free
1x tab
Protease Inhibitors
Make up to a final volume of 10ml
Volume of Stock
per 10ml
500 µl
300 µl
250 µl
1 ml
Stock
Concentration
1M
5M
10% w/v
10% w/v
Volume of Stock
per 10ml
500 µl
50 µl
Stock
Concentration
1M
0.2 M
40 µl
50 µl
n/a
250 mM
1M
n/a
2.3. Methods
2.3.1. Formaldehyde Crosslinking
2.3.1.1. Cell Culture
2.3.1.1.1. Adhesion Cells
Minimum of two plates are needed: one for colony counting (e.g. trypsin-EDTA treatment) and the other for
chromatin immunoprecipitation.
2.3.1.1.1.1.
2.3.1.1.1.2.
2.3.1.1.1.3.
2.3.1.1.1.4.
2.3.1.1.1.5.
Colony count one plate (e.g. cytometer) to estimate the colony number per plate; and determine
cell viability by staining (??????)
Add equivalent to 1% v/v formaldehyde directly into culture medium of the remaining plates
Note: Formaledhyde is highly toxic and all work must be done in a biosafety/fume cabinet
Incubate at RT for 10 min
Add equivalent to 125 mM Glycine to each plate
Incubate at RT for 5 min
Plate Volume
10 ml
15ml
2.3.1.1.1.6.
2.3.1.1.1.7.
2.3.1.1.1.8.
2.3.1.1.1.9.
2.3.1.1.1.10.
2.3.1.1.1.11.
2.3.1.1.1.12.
2.3.1.1.1.13.
2.3.1.1.1.14.
2.3.1.1.1.15.
2.3.1.1.1.14.
Formaldehyde Final
Concentration
1% v/v
1% v/v
Volume of 37% v/v
Formaldehyde
270 µl
405 µl
Glycine Final
Concentration
125 mM
125 mM
Volume of 1 M
Glycine
1.28 ml
1.93 ml
Asiprate medium from each plate
Add an equal volume, to that just aspirated, of ice-cold (4ºC) PBS onto each plate
Aspirate the PBS from each plate
Add 5 ml ice-cold (4ºC) PBS to each plate and scrap the cells using???????????? And transfer to
a 50ml conical falcon tube on ice
Rinse all the plates with 10 ml ice-cold (4C) PBS and pool with cells in the 50ml conical falcon tube
Centrifuge 50 ml conical falcon tube at 1,000xg(????) for 5 min at 4ºC
Place cells on ice and carefully aspirate the supernatant
Resuspend cells (equivalent to approximately 107 cells per ml) and dispense the cell suspension in
1.5 ml Tubes
Centrifuge the tubes at 12,000xg for 5 min at 4ºC
Return tubes to ice and carefully remove the supernatants
Aliquots can be either proceed to Cell Lysis (2.3.2.) or flash-frozen with liquid nitrogen and stored
at <-80C
2.3.1.1.2. Suspension Cells
2.3.1.1.2.1.
2.3.1.1.2.2.
2.3.1.1.2.3.
2.3.1.1.2.4.
2.3.1.1.2.5.
Culture Volume
10 ml
15ml
2.3.1.1.2.6.
2.3.1.1.2.7.
2.3.1.1.2.8.
2.3.1.1.2.9.
2.3.1.1.2.10.
2.3.1.1.2.11.
2.3.1.1.2.12.
2.3.1.1.2.13.
2.3.1.1.2.14.
2.3.1.1.1.15.
Colony count a sample from the culture (e.g. cytometer) and determine cell viability by staining
(??????)
Add equivalent to 1% v/v formaldehyde directly into culture medium
Note: Formaledhyde is highly toxic and all work must be done in a biosafety/fume cabinet
Incubate at RT for 10 min
Add equivalent to 125 mM Glycine to each plate
Incubate at RT for 5 min
Formaldehyde Final
Concentration
1% v/v
1% v/v
Volume of 37% v/v
Formaldehyde
270 µl
405 µl
Glycine Final
Concentration
125 mM
125 mM
Volume of 1 M
Glycine
1.28 ml
1.93 ml
Transfer the cell suspension to a 50 ml conical falcon tube
Centrifuge at 2,000xg for 5 min at 4ºC
Place cells on ice and carefully aspirate the supernatant
Add an equal volume, to that just aspirated, of ice-cold (4C) PBS
Centrifuge at 2,000xg for 5 min at 4ºC
Place cells on ice and carefully aspirate the supernatant
Resuspend cells (equivalent to 5x107 cells per ml) and dispense the cell suspension in 200 µl
aliquots (equivalent to 107 cells) in 1.5 ml Tubes
Centrifuge at 12,000xg for 1 min at 4ºC
Place cells on ice and carefully aspirate the supernatant
Aliquots can be either proceed to Cell Lysis (2.3.2.) or flash-frozen with liquid nitrogen and stored
at <-80ºC
2.3.1.2. Tissue
2.3.1.2.1. Crosslinked Tissues
2.3.1.2.1.1.
If applicable, thaw tissue sample on ice.
2.3.1.2.1.2.
2.3.1.2.2. Uncrosslinked Tissues
Tissue Grinder
2.3.2. Cell Lysis
If using a freshly prepared sample, proceed to 2.3.2.2.
If using a frozen sample, proceed to 2.3.2.1.
2.3.2.1.
2.3.2.4.
2.3.2.5.
2.3.2.6.
2.3.2.7.
Remove samples from the freezer and thaw on ice
Add 250 µl ice cold Cell Lysis Buffer per aliquot of 107 cells; resuspend by pipetting
Incubate on ice for 15 min
Centrifuge at 12,000xg for 2 min at 4ºC
Place cells on ice and carefully aspirate the supernatant and proceed to Nuclei Lysis (2.3.3.)
2.3.3. Nuclei Lysis
2.3.3.1.
2.3.3.2.
2.3.3.3.
2.3.3.4.
Resuspend nuclei extract in 200 µl cold (17C) Nuclei Lysis Buffer per 107 cells
Dispense 200 µl aliquots into 1.5 ml TPX tubes
Incubate on ice for 30 min
Proceed to Sonication (2.3.4.)
2.3.4. Sonication
2.3.4.1.
2.3.4.2.
2.3.4.3.
2.3.4.4.
2.3.4.5.
Turn on Bioruptor UCD-300 and Water Cooler and allow water temperature to reduce to 4ºC
Set Bioruptor USD 300 to High (“H”) power
Set Bioruptor to a repeated cycle of 10s ON and 20s OFF for 15 cycles
Secure samples into the Bioruptor USD 300 carousel and close the soundproof door
Run the sonication
Note: Maximum of consecutive 15 cycles
2.3.4.6. After every 15 cycles, allow the samples to cool for 2.5 min in the bioruptor
2.3.4.7. Remove samples from the sonicator
2.3.4.8. Pulse centrifuge the samples for 5 s
2.3.4.9. Repeated steps 2.3.4.6. to 2.3.4.9., if necessary, until material is correctly sonicated (see: SOP Diagenode
Bioruptor USD 300)
2.3.4.10. Centrifuge at 12,000xg for 10 min at 4ºC
2.3.4.11. Transfer supernatants into no-stick RNase/DNase-free 1.5 ml tubes
2.3.4.12. Resuspend debris pellet in 200 µl ic-cold 10 mM Tris-HCl, pH 8.0
2.3.4.13. Heavily vortex to resuspend debris pellet
2.3.4.14. Centrifuge at 12,000xg for 10 min at 4ºC
2.3.4.15. Pool the supernatants
2.3.4.16. Use a 20 µl sample to check the sonication (2.3.5.)
2.3.4.17. The bulk sample can be stored at -80ºC or proceed to Pre-Clearing (2.3.6.)
2.3.5. Checking Sonicated Samples
2.3.5.1.
2.3.5.2.
2.3.5.3.
2.3.5.4.
2.3.5.5.
2.3.5.6.
2.3.5.7.
2.3.5.8.
Add 68 µl ddH2O and 12 µl 5M NaCl to each sample (from 2.3.4.17.)
Using a hot block or PCR block, heat the samples to 100ºC for 20 min
Allow sample to cool to RT and then add 1 µl RNase Cocktail
Incubate sample, in a waterbath, at 37ºC for 30 min
Recover the DNA using a MinElute PCR Purification Kit; elute the DNA with 2x 10 µl EB buffer
Determine DNA concentration using a Nanodrop (see: ???)
Test 1-2 µg of DNA by DNA agarose gel electrophoresis (1.2% w/v agarose) (See: ????)
Correctly sonicated material (fragments between 200 to 500 bp), samples from 2.3.4.10. can proceed PreClearing (2.3.6.)
2.3.5.9 Incorrectly sonicated material (too large DNA fragments (>500 bp)), check Sonication Optimization Data for
the cell line and repool samples from 2.3.4.10. and proceed to Sonication (2.3.4.) for resonication
2.3.5.10 Incorrectly sonicated material (too small DNA fragments (<200 bp)), check Sonication Optimization Data for
the cell line and discard samples and start again from Cell Lysis (2.3.2.)
2.3.6. Pre-Clearing Sonicated Chromatin
2.3.6.1. Dispense 50 µl Dynalbeads (Protein A) into the required quantity of no-stick RNase/DNase-free 1.5 ml tubes
2.3.6.2. Place tubes into a magnetic rack and remove the supernatant
2.3.6.3. Add 250 µl 0.5% BSA/PBS to each tube
2.3.6.4. Remove the tubes from the magnetic rack and mix by vortex
2.3.6.5. Pulse centrifuge the samples for 5 s
2.3.6.6. Place tubes into a magnetic rack and remove the supernatant
2.3.6.7. Add 250 µl Chromatin Buffer H (Diagenode Auto-Histone Kit) to each tube
2.3.6.8. Remove the tubes from the magnetic rack and mix by vortex
2.3.6.9. Pulse centrifuge the samples for 5 s
2.3.6.10. Add the sonicated chromatin samples to the cleaned Dynalbeads
2.3.6.11. Roll-mix the samples at 4C for 1 hour
2.3.6.12. Pulse centrifuge the samples for 5 s
2.3.6.13. Place samples in the magnetic rack
2.3.6.14. Transfer the pre-cleared chromatin into no-stick RNase/DNase-free 1.5 ml tubes and proceed to Automated
Chromatin Immunoprecipitation (2.3.7.)
2.3.7. Automated Chromatin Immunoprecipitation
2.3.7.1.
2.3.7.2.
2.3.7.3.
2.3.7.4.
2.3.7.5.
2.3.7.6.
Remove Auto-ChIP kit from the refrigerator and allow all the solutions to equilibrate to RT
Turn on SX-8G IP-Star
Select [Protocols]
Select [ChIP]
Select [Indirect Method]
Select [ChIP _8_IPure_200_D.ptd] or [ChIP _16_IPure_200_D.ptd]
Note: 8 or 16: quantity of reactions (<8 = 8 and 8<x<16 = 16)
2.3.7.7. Enter amount of samples
2.3.7.8. Enter the following conditions:
IP Reaction:
10 hr
Beads Incubation:
2 hr
Washes:
5 min
2.3.7.9. Add all the indicated reagents and consumables; each highlighted block can be selected for more
information about the amount tips, PCR strips and reagents (name and volume).
Indirect Method (e.g. ChIP _16_IPure_100_D.ptd and 16 samples)





Two Full Tip Racks (e.g. 192)
Tips added to Reagent Tip Rack 1 (e.g. 12)
Reagents in Reagent Tip Rack 1 (e.g. A: DIB/Elution Buffer = 1700 µl and B to E:
IP Wash No. 1-4 = 1700 µl each)
Add reagents Large Reagent Rack (e.g. Beads Wash Buffer = 4200 µl)
PCR strips added with specified reagents (e.g. 16 strips with Tube 3: 10 µl
Magnetic Beads (10 µl beads = ~3 µg Antibody) and Tube 7: Sample (40 µl) +
Antibody (3 µg) + 1 µl 200x Proteinase Inhibitors (Diagenode) + Chromatin Buffer
H = 200 µl)
2.3.7.10. Close the door and review the procedure
2.3.7.10. Select [Run]
2.3.7.11. After the run, open the door as prompted
2.3.7.12. Remove all PCR strips; proceed to DNA Preparation (2.3.8.)
2.3.8. DNA Preparation
2.3.8.1.
2.3.8.2.
2.3.8.3.
2.3.8.4.
2.3.8.5.
2.3.8.6.
2.3.8.7.
2.3.8.8.
2.3.8.9.
Allow samples to equilibration to RT
Remove Chromatin Elution Buffer and 5M NaCl from the refrigerator and allow equilibration to RT
Remove Pre-cleared Sheared Chromatin from the -80C freezer and thawed on ice
Transfer 2 µl into the first PCR tube of the first row; termed the input
Add 94 µl Chromatin Elution Buffer and add 4 µl 5M NaCl to the input
Add 4 µl 5M NaCl to PCR tube 12 of every row
Incubate the PCR strips at 65ºC for 4 hours or overnight
Pulse centrifuge the PCR tubes for 5s
Insert PCR strips into the Magnetic PCR Tube Rack and carefully pipette off each sample supernatant, in
PCR tube 12 and the input, into No-Stick Dnase-Free 1.5 ml Tubes
2.3.8.10. Add 2 µl RNAse Cocktail to the input and samples
2.3.8.11. Incubate input and samples in a waterbath at 37ºC for 30 min
2.3.8.12. Add 2 µl Proteinase K to the input and samples
2.3.8.13. Incubate input and samples in a waterbath at 65ºC for 30 min
2.3.8.14. Purify the DNA from each input and sample using a MinElute PCR Purification Kit; elute with two times 10 µl
ddH2O steps
2.3.8.15. The purified DNA for the samples and input DNA can either proceed to DNA Analysis (2.3.9.) or stored at
<-20ºC
2.3.9. DNA Analysis
2.3.9.1. Test 1 µl of each sample, and input DNA, on the Bioanalyzer (See: SOP Bioanalyzer)
Note: If Bioanalyzer shows very little or incorrectly fragmentation of DNA (majority <200 bp and >500 bp),
then the procedure will have to be started again from 2.3.2.
2.3.9.2. Test 1 µl of each sample and input DNA by qPCR (see: SOP qPCR) using relevant primers to the antibody
and cell line
Note: If qPCR does not show enrichment of target DNA, over the controls, then it is assumed that the
chromatin immunoprecipitation was unsuccessful; the process then must be reviewed and started again
from 2.3.2.
2.3.9.3. Use DNA from samples and input DNA to make Illumina Libraries (see: SOP Illumina Library Construction)
2.3.9.4. Test 2 ng of each library qPCR (see: SOP qPCR) using relevant primers to the antibody and cell line
Note: Do not proceed with Next Generation Sequencing, unless there is a 20 fold enrichment of the target
genes in comparison to the controls
2.3.9.5. Sequence the libraries using Illumina HiSeq 2000 (See: SOP Illumina HiSeq 2000)
3. Records
4. Associated Documents