Instrument: Illumina HiSeq 4000
Strategy: ChIP-Seq
Source: GENOMIC
Selection: ChIP
Layout: SINGLE
Construction protocol: Cells were washed twice with ice-cold PBS, scraped, centrifuged for 10 min at 4000 rpm, resuspended in cell lysis buffer (5 mM PIPES, pH 8.0, 85 mM KCl, and 0.5% NP-40, 1 ml/15 cm plate) and incubated for 10 min on ice. During the incubation, the lysates were repeatedly pipetted up and down every 5 minutes. Lysates were then centrifuged for 10 min at 4000 rpm. Nuclear pellets were resuspended in 6 volumes of sonication buffer (50 mM Tris-HCl, pH 8.1, 10 mM EDTA, 0.1% SDS), incubated on ice for 10 min, and sonicated to obtain DNA fragments below 2000 bp in length (Covaris S220 sonicator, 20% Duty factor, 200 cycles/burst, 100 peak incident power, 50 cycles of 30" on and 30" off). Sonicated lysates were cleared by centrifugation and 400-1600 μg of chromatin was diluted in RIPA buffer (10 mM Tris-HCl, pH 8.0, 1 mM EDTA, 0.5 mM EGTA, 1% Triton X-100, 0.1% SDS, 0.1% Na-deoxycholate, 140 mM NaCl) to a final concentration of 0.8 μg/μl, precleared with Protein A sepharose (GE Healthcare) for 2 hours at 4°C and immunoprecipitated overnight with 8-16 μg of normal rabbit IgGs, anti-Rad21 or anti-CTCF antibodies. About 15% of the precleared chromatin was saved as input. Immunoprecipitated DNA was purified with the Qiagen QIAquick PCR Purification Kit, eluted in 60 μl of water and analyzed by qPCR together with 2% of the input chromatin prior to ChIP-Seq library preparation. ChIP-seq libraries were prepared independently from two ChIP biological replicates using the Illumina TruSeq™ DNA sample preparation kit according to manufacturer instructions with few modifications. We used 100 ng of ChIP input DNA (as measured by Fragment analyzer™) and 50 μl of immunoprecipitated DNA as a starting material; Illumina adapters were diluted 1:50, and library samples were enriched through 18 cycles of PCR amplification. We assessed library quality and fragment size by qPCR and Fragment analyzer™, and when necessary we performed an additional size selection step on agarose gel after PCR amplification to enrich for fragments between 150 and 500 bp. We sequenced 4 to 8 multiplexed libraries per lane on the Illumina HiSeq4000 sequencing platform (single end-reads, 50 bp long) at the Vincent J. Coates Genomics Sequencing Laboratory at UC Berkeley, supported by NIH S10 OD018174 Instrumentation Grant.