ChIP-Sequencing

Adapted from Myers Lab ChIP-seq Protocol v011014

Johnson et al., 2007, Science 316: 1497- 1502

Reagents:

• cOmplete protease inhibitor cocktail pills (Sigma # 11697498001)

• Large TC Dish 245x245x25 (ThermoFisher #166508)

• DMEM, powder, high glucose, pyruvate (ThermoFisher #12800017)

• Dynabeads M-280 Sheep anti-Mouse IgG (Invitrogen #11201D) or Dynabeads M-280

  Sheep anti-Rabbit IgG (Invitrogen #11203D)

• DynaMag-2 (Life Technologies #12321D)

• BSA (Fisher #BP1605-100G)

• QIAquick PCR Purification Kit (Qiagen #28104)

• DNA LoBind Tube (Eppendorf #022431021)

• Qubit™ dsDNA HS Assay Kit (ThermoFisher #Q32851)

Buffers:

*Autoclave solutions to sterilize

Tricine-buffered saline (TBS) (store at RT)

137 mM NaCl

5 mM KCl

0.5 mM MgCl

0.7 mM CaCl

25 mM Tricine

Adjust pH to 7.35 with 10 N NaOH

1x Phosphate-buffered saline (PBS) (store at RT)

137mM NaCl

2.7 mM KCl

10 mM Na2HPO4

1.8 mM KH2PO4

Adjust pH to 7.2 with 10 N NaOH

1x TE (store at RT)

10 mM Tris

1 mM EDTA

*Sterile filter solutions with 0.2um filter

Quenching Solution (store at RT)

2.5 M Glycine

Farnham Lysis Buffer (FLB) (store at 4 degrees)

5mM PIPES (Piperazine-1,4-bis(2-ethanesulfonic acid)) pH 8.0

85mM KCl

0.5% IGEPAL (N3500 Nonidet-P40 substitute CAS #9036-19-5)

RIPA Buffer (store at 4 degrees)

1xPBS

1% IGEPAL (N3500 Nonidet-P40 substitute CAS #9036-19-5)

0.5% Sodium Deoxycholate

0.1% SDS

+Protease inhibitors the day of use

LiCl IP Wash Buffer (store at 4 degrees)

100mM Tris-HCl pH 7.5

500mM LiCl

1% NP-40

1% Sodium Deoxycholate

BSA/PBS (store at 4 degrees)

5mg/mL BSA

1x PBS

Infection:

*In 245x245x25 tissue culture plates there are approximately 7 x 107 MRC5 cells at confluence.

Make sure to plate and grow cells at 37℃ until they are completely confluent

To infect at an MOI of 10 infect with 108 PFU/mL, 7mL/plate

• Dilute appropriate virus in cold TBS

• Aspirate media from plates

• Add on 7 mL of viral inoculum

• Adsorb for 1 hour at room temperature (RT), rock gently every 10 minutes

• Prep media and TBS, and place in 37℃ water bath to prewarm

  ○Prep 50 mL 2% FBS 1X DMEM media per plate

  ○Prep 50 mL TBS per plate

• Aspirate viral inoculum

• Wash with 50 mL prewarmed TBS

• Add 50 mL prewarmed 2% FBS 1X DMEM media

• Incubate at 37℃ until harvest

Cross-linking and harvesting:

*All future steps should be performed on ice, and all solutions should be kept chilled.

• Prep solutions day of use:

  ○25% formaldehyde

    ▪Dilute stock 36.5% formaldehyde in water

    ▪Need 5mL per plate

  ○FLB + 1x Protease Inhibitor (PI) pills

    ▪1 PI pill dissolved per 50 mL solution

    ▪Need 50mL solution per plate

• Add 5 mL 25% formaldehyde to plate, swirl to mix

• Incubate RT 10 min

• Add 5mL quenching solution (2.5 M glycine), swirl to mix

• Aspirate medium from plates

• Add 50 mL cold TBS

• Aspirate wash completely

• Add 50 mL cold FLB + PI to each plate

• Scrape cells using large scraper into 50 mL conical

• Spin at 2500 rpm 10 min at 4℃

• Aspirate supernatant (SN)

*If necessary can flash freeze pellet in liquid nitrogen and store at -80℃ until on Day 2 of the

protocol. Alternatively can aspirate SN and immediately add 1.1mL RIPA+PIs, starting on step 4

of the Day 2 protocol.

Day 1

Equilibrate beads:

• Combine 50 μL Dynabeads M-280 Sheep anti-Mouse or anti-Rabbit magnetic beads with 1mL 5mg/mL BSA in 1x PBS

• Place on Dynamag-2 magnet for 2 minutes, aspirate SN

• Wash with 1mL 5 mg/mL BSA in PBS (x3)

• Resuspend in 1 mL BSA/PBS

Bind primary antibody to beads:

• Add 5-25 μg primary antibody to magnetic beads

• Rotate overnight at 4℃

Day 2

Sonication:

• Prep solutions day of use:

  ○FLB + 1x Protease Inhibitor (PI) pills

   ▪1 PI pill dissolved per 50 mL solution

   ▪Need 50mL solution per plate

  ○1x RIPA 1x PI's

   ▪1 PI pill dissolved per 50 mL solution

   ▪Need 1.1mL solution per plate

• Thaw nuclear pellets on ice.

• Resuspend pellet gently in 4mL FLB + PI (invert tube to dislodge pellet, want to keep the pellet intact!)

• Spin 2500rpm 10min 4℃, aspirate SN

• Resuspend pellet gently in 1.1mL RIPA + PI. (Again do not breakup pellet, just dislodge from tube).

• Transfer pellet into 1.5mL microfuge tube (I use a 2mL pipette to suck up the intact pellet).

• Pipette up and down or vortex to break up pellet.

• Sonicate 6 x 30sec, power 40%, do in ice bath (3 min total pulse time)

  ○Ensure solution is homogenous without any fragments floating

• Spin 14000rpm 4℃ 15min

• Aliquot 50uL SN into screwtop tube and place at -80℃ to use as “input”

• Split remaining volume of nuclear lysate equally for the number of IP’s per sample

  ○For two antibodies: aliquot 500uL of each sample

Immunoprecipitation:

• Wash beads from Day 1 with 1mL 5 mg/mL BSA/PBS (x3)

• Resuspend beads in final volume of 100uL 5 mg/mL BSA/PBS

• Add 100 μL beads to each nuclear lysate sample

• Rotate overnight at 4℃

Day 3

Wash beads

• Prep solutions day of use:

  ○2x IP Elution Buffer

   ▪2% SDS

   ▪0.2M NaHCO3

  ○1x IP Elution Buffer

   ▪1% SDS

   ▪0.1M NaHCO3

• Place microfuge tubes on magnet for 2 min and aspirate SN

• Wash with 1mL LiCl wash buffer 3 min rotating 4℃ (x7)

• Wash with 1mL 1x TE 1 min, place on magnet

Elute beads

• Aspirate SN, resuspend in 200uL 1x IP elution buffer. Vortex well

• Incubate at 65℃ 900rpm 2hrs

• Spin 3min 12000 rpm RT, magnet. Transfer SN to screwtop tube.

• Add 50uL 1x TE + 100uL 2x IP elution buffer to the 50uL aliquot of input.

• Incubate Input and IP Chromatin at 65℃ 900 rpm O/N

Day 4

Extract and purify DNA

• Add equal volume (200uL) phenol:chloroform:isoamyl alcohol (25:24:1) to samples

• Spin 12000 rpm 15 min RT

• Transfer top, aqueous phase to new tube

• Back extract: add 50uL water to lower phase in original tube

• Spin 12krpm 15 min RT

• Combine top, aqueous phase with previously extracted phase

• Add equal volume (200uL) chloroform:isoamyl alcohol (24:1) to samples

• Extract with 200uL Chloroform:Isoamyl alcohol (24:1)

• Spin 12krpm 5 min RT

• Transfer top, aqueous phase to new tube

• Purify with Qiagen PCR cleanup column

  ○In first step make sure to add 10uL NaOAc pH 5.0

  ○Elute with 60uL buffer EB into DNA LoBind Tube

• Measure DNA using Qubit 2.0 and the Qubit™ dsDNA HS Assay Kit

Library Synthesis

Reagents:

• NEB Ultra II DNA Library Prep Kit w/ Beads (NEB #E7103S)

• NEBNext^® Multiplex Oligos for Illumina® (Index Primers Set 1) (NEB #E7335S)

• Magnetic Stand-96 (Ambion RNA by Life Technologies #1111049)

• Multiplate PCR Plates 96-well, clear (Bio-Rad #MLP9601)

• Microseal 'B' seal Seals (Bio-Rad #MSB1001)

• Agilent DNA 7500 Kit Agilent # 5067-1506

Follow NEBNext^® Ultra™ II DNA Library Prep Kit for Illumina instructions (manualE7645)

• Generally use 20 ng Input DNA, and up to 20 ng of IP DNA

• Perform size selection (Step 3A), selecting for approximate final library size of 270 bp

• Assess concentration and purity using Agilent DNA 7500 Kit (Agilent #5067-1506)