title: GSM5543440: Input DNA; Rattus norvegicus; ChIP-Seq
library_construction_protocol: 40 million INS1 832/13 cells were cross-linked with 4% formaldehyde for 10 min, followed by quenching with 125 mM glycine for 5 min at room temperature. Fixed cells were washed with 1X TBS and kept at -80oC. Briefly, about 40 million cells were resuspended in 2 ml of cell lysis buffer (10 mM Tris-HCl, pH7.5, 10 mM NaCl, 0.5% NP-40) and incubated on ice for 10 min. The lysates were washed with MNase digestion buffer (20 mM Tris-HCl, pH7.5, 15 mM NaCl, 60 mM KCl, 1 mM CaCl2) and incubated with 3,000 or 5,000 gel units of MNase (M0247S, New England Biolabs, Ipswich, MA) for 20 minutes at 37 °C. After adding the same volume of sonication buffer (100 mM Tris-HCl, pH8.1, 20 mM EDTA, 200 mM NaCl, 2% Triton X-100, 0.2% Sodium deoxycholate), the lysate was sonicated for 3.5 min (30 sec-on / 30 sec-off) in Diagenode bioruptor and centrifuged at 15,000 rpm for 10 min. The cleared supernatant equivalent to about 40 million cells was incubated with 2.5 µg of anti-DBP antibody (PM079, MBL Life Science, Woburn, MA) previously validated for ChIP-seq. Subsequent immunoprecipitation and library preparation were performed as described (Zhong, J. et al, BMC Genomics, 2017).  The libraries were sequenced to 51 base pairs from both ends on an Illumina NextSeq instrument (Illumina) according to manufacturer's instructions.