GSM6318210: Hela, treated with CBP30, input chromatin; Homo sapiens; ChIP-Seq
Cells were then lysed under hypotonic conditions in 1 mL of 20 mM Tris-HCl pH 8, 85 mM KCl, 0.5% NP- 40 (1 tablet of protease inhibitor (Roche, 11836170001) per 5 mL of buffer) for 30 min on ice. Nuclei (and other insoluble material) were collected by centrifugation at 1300 x g for 5 min at 4 degC, lysed by resuspension in 150 uL 50 mM Tris-HCl pH 8, 150 mM NaCl, 2 mM EDTA, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS (1 tablet of protease inhibitor per 5 mL of buffer), and passaged five times through a 27-gauge needle (BD #309623 Lot: 0227218). Lysate was then diluted to 500 uL by addition of 350 uL of binding buffer (25 mM HEPES pH 7.5, 100 mM NaCl, 0.1% NP-40).  Five uL of RNAse A/T1 (Thermo Scientific, EN0551) was added, and the sample was incubated at 37 degC for 25 min. Next, CaCl\textsubscript{2} was added to a final concentration of 40 mM (21 uL of 1 M) followed by the addition of 75 U (3 uL of 25 U/uL) of micrococcal nuclease (MNase, Worthington Biochemical) and incubated at 37 degC for 5 min. MNase was quenched by the addition of 40 mM EDTA (46 uL of 500 mM EDTA), and the total volume was brought to 1 mL by the addition of 425 uL of binding buffer. Next, insolubilities were removed by centrifugation at max speed (about 21,000 x g) at 4 degC for 5 min, and the supernatant containing soluble chromatin was collected. Library preparation was done using KAPA HyperPrep Kit (Roche, KK8504) with 4 uL of Illumina adapters (IDT, UDI) and sequenced on Illumina NextSeq 500 with a Mid-Output (paired-end 75 bp reads) flow cell. Input libraries had between 83-102M reads, and each IP library had 27-46M reads that passed QC, with 88% of the bases having quality scores >= 30.