GSM5573438: 786-O A31_rV5_IP; Homo sapiens; ChIP-Seq
Cell pellets were thawed and resuspended in 2 ml of cell lysis buffer (5 mM PIPES, pH 8.0, 85 mM KCl, and 0.5% NP-40, 1 ml/15 cm plate) w/ protease inhibitors and incubated for 10' on ice. Lysates were centrifuged for 10' at 4000 rpm and nuclear pellets resuspended in 6 volumes of sonication buffer (50 mM Tris-HCl, pH 8.1, 10 mM EDTA, 0.1% SDS) w/ protease inhibitors, incubated on ice for 10', and sonicated to obtain DNA fragments around 500 bp in length (Covaris S220 sonicator, 20% Duty factor, 200 cycles/burst, 150 peak incident power, 10 cycles 30” on and 30" off). Sonicated lysates were cleared by centrifugation and chromatin (400 ug per antibody) 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) w/ protease inhibitors to a final concentration of 0.8 ug/ul. ChIP-seq libraries were prepared using the NEBNext® Ultra™ II DNA Library Prep Kit for Illumina® (NEB E7645) according to manufacturer instructions with a few modifications. 20 ng of ChIP input DNA (as measured by Nanodrop) and 25 ul of the immunoprecipitated DNA were used as a starting material and the recommended reagents' volumes were cut in half. The NEBNext Adaptor for Illumina was diluted 1:10 in Tris/NaCl, pH 8.0 (10 mM Tris-HCl pH 8.0, 10 mM NaCl) and the ligation step extended to 30'. After ligation, a single purification step with 0.9X volumes of Agencourt AMPure XP PCR purification beads (Beckman Coulter A63880) was performed, eluting DNA in 22 ul of 10 mM Tris-HCl pH 8.0. 20 ul of the eluted DNA were used for the library enrichment step, performed with the KAPA HotStart PCR kit (Roche Diagnostics KK2502) in 50 ul of total reaction volume (10 ul 5X KAPA buffer, 1.5 ul 10 mM dNTPs, 0.5 ul 10 uM NEB Universal PCR primer, 0.5 ul 10 uM NEB index primer, 1 ul KAPA polymerase, 16.5 ul nuclease-free water and 20 ul sample). Samples were enriched with 9 PCR cycles (98 'C, 45”; [98 'C, 15”; 60 'C, 10”] x 9; 72 'C, 1'; 4 'C, hold), purified with 0.9 volumes of AMPure XP PCR purification beads and eluted with 33 ul of 10 mM Tris-HCl pH 8.0. Library concentration, quality and fragment size were assessed by Qubit fluorometric quantification (Qubit™ dsDNA HS Assay Kit, InvitrogenTM Q32851) qPCR and Fragment analyzer™. Libraries were pooled and sequenced in one lane on the Illumina HiSeq4000 sequencing platform (50-bp, single end-reads).