alias: GSM3681604
title: GSM3681604: Nascent_ChORseq_RNApolIIS5phos_rep2; Mus musculus; OTHER
library_construction_protocol: Fixed cells were lysed for 20 minutes in ice-cold lysis buffer (100mM NaCl, 66mM Tris-HCl pH 8, 5mM EDTA, 0.3% SDS, 1.6% Triton X-100) supplemented with protease inhibitors, passed through a 21G needle, and sonicated using a Covaris M220 (Thermo Fisher) to obtain chromatin fragment sizes of 250-500 bp. Sonicated chromatin was centrifuged at 14,000 rpm at 4ºC for 10 minutes and the supernatant used for subsequent steps. In parallel, and independently, Drosophila S2 cells were fixed, lysed and sonicated as described above. After sonication, mESC input chromatin required for the analysis of all time-points from the same time course experiment was mixed with Drosophila S2 chromatin (2.5% of total chromatin). Chromatin was immunoprecipitated by incubating sonicated chromatin with antibody (Abcam, ab5131). Libraries were constructed using the KAPA Hyperprep kit following manufacturer's instructions, except that 1.25 µM of Illumina-compatible indexed adapters (Pentabase) were ligated to A-tailed DNA. Following adapter ligation, biotin-TEG-azide (Berry&Associates) was used to isolate EdU-labelled DNA via Click-iT (Invitrogen). Biotinylated products were captured using MyOne Streptavidin T1 beads (Thermo Fisher Scientific) and subjected to PCR amplification following the KAPA Hyperprep kit protocol.
identifiers.namespace: GEO
identifiers.id: GSM3681604