GSM9453401: H3K27me3, AM, REP1; Mus musculus; ChIP-Seq
Frozen tissue was sent to Active Motif Services (Carlsbad, CA) for ChIP-Seq. Active Motif prepared chromatin, performed ChIP reactions, generated libraries, sequenced the libraries and performed data analysis. In brief, tissue was submersed in PBS + 1% formaldehyde, cut into small pieces and incubated 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, followed by disruption with a Dounce homogenizer, washing and sonication using the PIXUL® Multi-Sample Sonicator (Active Motif, Catalog #53130) to shear DNA to an average fragment size of 200–1000 bp. To determine chromatin yield, an aliquot of the sheared chromatin was reverse crosslinked at 65°C, treated with RNase and proteinase K, and subjected to DNA purification using SPRI beads (Beckman Coulter). DNA concentrations were measured using a Qubit Fluorometer (Thermo Fisher), and total chromatin yield was extrapolated based on the original chromatin volume. 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.