GSM5987579: NCoR1_control_RNA-seq_rep2; Mus musculus; RNA-Seq
RNA-seq RNA was isolated from snap-frozen BAT as previously described (23) using TRIzol-chloroform extraction followed by RNeasy Mini spin columns (Qiagen) and on-column DNase-digestion Novogene or TruSeq ChIP-seq Chromatin immunoprecipitation (ChIP) was done as previously described with minor modifications (23). Total protein for each sonicated sample was quantified by BCA and normalized to 1.5mg of protein for each ChIP prior to incubation with antibody overnight. 5% input was saved prior to antibody incubation and used as input control. Antibodies used were HDAC3 (Genetex GTX109679) and rabbit IgG (CST 2729S) and immunoprecipitated with BSA blocked Protein A agarose beads (Invitrogen 15918014). ChIP DNA was isolated using phenol/chloroform extraction and treated with RNAse A prior to sequencing library preparation. The NEBNext Ultra II DNA Library Prep Kit for Illumina (NEB #E7645S) with AMPure XP Beads (Beckman Coulter, Inc. #A63881) was used to amplify and size select ChIP DNA. Agilent High Sensitivity DNA Kit (5007-4626) KAPA Library Quantification Kit (KR0405) were used to quantify libraries and fragment sizes prior to sequencing. An n=3 was sequenced for NCoR1/2 Control and NCoR1/2 BAT-dKO groups, n=2 for HDAC3 BAT-KO, and n=1 each for input and IgG. The sequencing was done at the Next-Generation Sequencing Core at the University of Pennsylvania on a NextSeq platform (Illumina) at 100SR for a total of 30 million reads per sample.