GSM9170950: Total chromatin (pooled input); Mus musculus; ChIP-Seq
Tissue was pulverized with mortar and pestle in liquid nitrogen and then fixed in PBS + 1% formaldehyde at room temperature for 15 minutes. Fixation was stopped by the addition of 0.125 M glycine (final). Chromatin was isolated by adding lysis buffer and sonicating using the PIXUL® Multi-Sample Sonicator (Active Motif, Catalog #53130) to shear DNA to an average fragment size of 200–1000 bp. Aliquots of chromatin were precleared with protein G agarose beads (Invitrogen). Immunoprecipitations were performed using an antibody specific to PGC-1α (Novus NBP1-04676). After washing, immune complexes were eluted from the beads using SDS buffer, treated with RNase and proteinase K, and de-crosslinked by overnight incubation at 65°C. ChIP DNA was then purified using phenol-chloroform extraction and ethanol precipitation. ChIP DNA libraries were prepared using either the PrepX DNA Library Kit (Takara Bio) on the Apollo automation platform or the NEB DNA Library Prep Kit, following the manufacturers' protocols. Libraries were sequenced on the Illumina platform and the resulting data were analyzed using standard ChIP-Seq workflows.