GSM4727158: U2OS_HYPER_PolII-2SP; Homo sapiens; ChIP-Seq
ChIP-seq experiments were carried out using the HighCell# ChIP-Protein G kit (Diagenode, Cat#:  C01010063) following the manufacturer's protocol. Chromatin from five million cells was used per ChIP reaction with 6.5 μg of the target protein antibody. In brief, cells were introduced to isotonic (150 mM Na+) or hypertonic (300 mM Na+) conditions for 30 mins, trypsinized in solutions that maintained tonicity and washed twice with 1x PBS (isotonic) or 2 x PBS (hypertonic). This was followed by cross-linking for 12 min in 1% formaldehyde solution in iso or hyper-osmotic PBS (Sigma-Aldrich, Cat#: F8775-25ML). Crosslinking was terminated by the addition of 1/10 volume 1.25 M glycine for 5 min at room temperature. This was followed by cell lysis and sonication (Bioruptor, Diagenode) resulting in an average chromatin fragment size of 200 bp. Fragmented chromatin was used for immunoprecipitation using various antibodies with an overnight incubation at 4 °C. Immunoprecipitated DNA was de-crosslinked and purified using the iPure Kit V2 (Diagenode, Cat#: C03010015) using the manufacturer's protocol. Purified DNA was quantified using Qubit-3 (Invitrogen) and 10-100 ng of total DNA was used for library preparation using the KAPA Hyper Prep kit (KAPABiosystems, Cat#: KK8500) as per the protocol. Briefly, DNA was first converted to blunt-ended fragments via end-repair and a single 'A' nucleotide was added to fragment ends. This was immediately followed by ligation of Illumina adaptors and PCR amplification (for 9-12 cycles) using Illumina barcoding primers and Phusion DNA polymerase (NEB, Cat# M0530S). PCR products were double size-selected using the KAPA Pure beads (Roche, Cat#: 07983280001) to remove fragments larger than 500 bp (0.65X beads:sample ratio) and primer dimers (0.9X beads:sample ratio). In the end, all libraries were quantified and quality checked using the Bioanalyzer 2100 (Agilent) and sequenced on the Illumina HiSeq 2500 Sequencer (paired-end, 50-nucleotide read length) to roughly 30-40 million total reads.