alias: GSM2889136
title: GSM2889136: Cold brown adipocyte H3K27ac ChIP rep 1; Mus musculus; ChIP-Seq
library_construction_protocol: Tissues were dounce homogenized, and cross-linked nuclei were isolated and sorted by BD FACS Aria II using mCherry fluorescence. Sorted nuclei were sheared by Covaris E220 and used for ChIP for overnight. Immunoprecipitates were washed and subjected to elution and reverse crosslinking. DNA was then extracted by AMPure XP beads according to the manufacturer's manual. Sequencing libraries were constructed using the “on-bead” preparation method as previously described (Roh et al., Cell Rep, 2017). ChIP-isolated DNA was subjected to end repair/phosphorylation using the End-It DNA End-Repair Kit (Epicentre), A-tailing using the Klenow Fragment and index adaptor ligation using Quick Ligase (NEB). AMPure XP beads were left in all the reactions to allow isolation of DNA using PEG (Polyethylene Glycol 8000)/NaCl solution, thus minimizing loss between reactions. After ligation, DNA was eluted and amplified by PCR for 14-16 cycles using the PfuUltra II Hotstart PCR Master Mix. DNA sized between 250 and 600bp was selected by gel electrophoresis and extraction using E-Gel EX Agarose Gels (Invitrogen) and MinElute Gel Extraction kits (Qiagen). Libraries were analyzed using the Qubit and Agilent Bioanalyzer, pooled at a final concentration of 12pM and sequenced on a HiSeq2500 or NextSeq500 system.
identifiers.namespace: GEO
identifiers.id: GSM2889136