Instrument: NextSeq 500
Strategy: ChIP-Seq
Source: GENOMIC
Selection: ChIP
Layout: SINGLE
Construction protocol: Cells were grown to 80% confluence on 150cm plates and washed once with PBS and fixed with 1% formaldehyde (Thermo Fisher #28908) in 10 ml PBS for 10 min. To quench the cross-linking reaction, 1/20 volume of 2.5 M glycine was added to the plate for a final concentration of 0.125M for 5 min. The supernatant was aspirated, and the cells were washed twice with 5 ml PBS and harvested on ice using cell scrapers into 15ml sonication tubes (Diagenode cat# C01020031). Cells were pelleted at 1000g for 5 min at 4°C, washed 1x with 5 ml cold PBS, and pelleted once more. Cell pellet collected from 6 plates of cells was resuspended in 3 ml of Lysis Buffer 1 (50 mM HEPES-KOH, pH 7.5; 140 mM NaCl, 1mM EDTA, 10% glycerol, 0.5% NP-40, 0.25% Triton X-100), allowed to rotate for 10 min at 4°C, and pelleted at 1000 x g for 5 min at 4°C. Cells were then resuspended in 3 ml of Lysis Buffer 2 (10 mM Tris-HCL pH 8.0, 200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA), allowed to rotate for 10 min at room temperature, and pelleted at 1000 x g for 5 min at 4°C. Cells were then resuspended in 3 ml of Lysis Buffer 3 (10 mM Tris-HCl pH 8, 100 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 0.1% Na-Deoxycholate, 0.5% N-lauroylsarcosine) and then sonicated on a Bioruptor Pico at 4°C for 20min of 30'' ON-OFF cycles with 1.2 g of sonication beads. After sonication, samples were transferred to 2 separate 1.5 ml microtube and centrifuged at 20,000 x g for 15 minutes at 4°C. The supernatant was transferred to new 15 ml tubes. To check sonication efficiency, 20µl of sonicated samples was transferred to a new microtube with 80µl PBS and incubated at 65°C for 3hr on an Eppendorf Thermomixer shaking at 1000rpm to decrosslink. DNA was purified with the QIAquick PCR Purification Kit (Qiagen cat# 28106) and eluted in 50µl EB buffer. 1 ug of decrosslinked chromatin from each sample were run in a 1% agarose gel at 100V for 45min and imaged on a Bio-rad ChemiDoc XRS+. DNA concentration in the sonicated decrosslinked samples were measured via qubit, and this concentration was then used to estimate the concentration of chromatin in the un-decrosslinked samples. 30 ug of total chromatin and 50 ng of Spike-in chromatin (Active Motif 53083) weres transferred to a 1.5ml LoBind tube (Eppendorf cat# 0030108051) and brought up to 500µl final volume with Lysis Buffer 3. 5µl was removed as input material (1%) and placed in a separate microtube at 4°C. 2µg antibody was used for each histone ChIP, and 5ug was used for transcription factor ChIP (see Key Resources table for list of antibodies) along with 1ug of Spike-in antibodies (Active Motif 61686). Antibodies were pre-bound to 50 ul of Dynabeads Protein G (Thermo Fisher 10004D), which was washed in 1 ml of 0.5% BSA in 1x PBS three times before and after overnight pre-binding at 4°C. The chromatin samples were added to the pre-bound Dynabeads and rotated end-to-end overnight at 4°C. Beads were washed 5x with 1 ml Wash Buffer (50 mM Hepes-KOH, pH 7.6; 500 mM LiCl, 1 mM EDTA, 1% NP-40; 0.5% Na-Deoxycholate) and once with 1 ml TE containing 50 mM NaCl. Beads were dried after the last wash by removing as much TE as possible. 210 ul of Elution Buffer (50 mM Tris-HCl, pH 8; 10 mM EDTA, 1% SDS) was added to each sample and 45 ul Lysis Buffer 3 plus 150 ul elution buffer were added to each input sample that was previously set aside. To elute immunocomplexes from beads, samples were incubated at 65°C for 15 minutes on an Eppendorf Thermomixer shaking at 1000rpm. Tubes were centrifuged for 3min at 2000rpm at RT and 200µl of supernatant was transferred to a new tube and reverse crosslinked overnight at 65°C. Next day, 200 ul of TE was added to each decrosslinked sample, and the samples were treated sequentially with 0.2 mg/ml RNAse A (Sigma R5503) at 37°C and 0.2 ug/ml Proteinase K (NEB P8107S) at 55°C for two hours each. DNA purification was performed with the QIAquick PCR Purification Kit (Qiagen cat# 28106) and eluted in 50µl EB buffer and quantified by Qubit. Immunoprecipitated DNA was used to generate libraries using the NEBNext Ultra DNA Library Prep Kit for Illumina (New England Biolabs cat# 7370) following the manufacturer's instructions.