title: GSM4874314: input_ESC_DMSO_Pol2; Mus musculus; ChIP-Seq
library_construction_protocol: Cells were detached with TrypLE, washed once with PBS, and protein-protein crosslinking was performed by incubating cells in rotation with 2 mM disuccimidyl glutarate in PBS for 45 min in room temperature. Cells were washed twice with PBS and DNA-protein crosslinking was performed with 1 % formaldehyde for 10 minutes in room temperature followed by quenching with 125 mM glycine. For each sample, 30 million mESCs (and an exogenous genome spike-in of 7.5 million S2 cells) were lysed in LB1 (50 mM HEPES-KOH, 140 nM NaCl, 1 mM EDTA, 10 % glycerol, 0.5 % Igepal ca-630 and 0.25 % Triton X-100, 5 mM Na-butyrate and 1x protease inhibitor cocktail) and collected by sentrifugation. Lysis was continued in LB2 (10 mM Tris-HCl pH 8.0, 200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 5 mM Na-butarate and 1 x protease inhibitor cocktail) followed by sentrifugation, nuclei were lysed in sonication buffer (50 mM HEPES-KOH pH 7.5, 140 mM EDTA, 1 mM EDTA, 1 mM EGTA, 1 % Triton X-100, 0.1 % Na-deoxycholate, 0.1 % SDS and 1x protease inhibitors (Roche)) and sonicated for 50 cycles by using Bioruptor NextGen (high setting). Ten percent of sample material was set aside as input and stored in +4C. Voume was adjusted to 200 µl with elution buffer (50 mM Tris-HCl pH 8.0, 10 mM EDTA and 1% SDS) and crosslinks were reversed by incubating samples in 65C for 16 hours. Samples were topped up with 200 µl TE buffer and 8 µl of RNAse A, incubated in 37 C for 2 hours followed by addition of 7 µl of 300 mM CaCl2, 2 µl Proteinase K (Ambion) and 30 minutes incubation in 37C. DNA purified from samples with phenol:chloroform:isoamyl alcohol extraction followed by ethanol precipitation. 10 ng of DNA was used to prepare sequencing libraries with KAPA HyperPrep Kit (Roche) according to manufacturer's instructions.
attributes.tag: GEO Accession
attributes.value: GSM4874314