GSM8841763: 19_1st_ChIPseq_shGFP_NCOR_rp1; Mus musculus; ChIP-Seq
Fresh cells were cross-linked with 2 mM disuccinimidyl glutarate (DSG) for 30 minutes, followed by 1% formaldehyde for 10 minutes at room temperature. Nuclei were isolated, sonicated, and incubated with magnetic bead-antibody complexes.  Antibodies to NCOR (bethyl, A301-145A), SMRT (bethyl, A301-147A), and GPS2 (homemade) were used. ChIP-Seq: ChIPed DNA was processed using Takara SMRTer ThruPLEX DNA-seq Kit. Briefly, DNA was end-repaired and ligated to stem-loop adaptors. Then DNA was PCR amplified with Illumina-compatible primers for 7-11 cycles and library fragments of 200 to 400 bp were selected using Ampure XP beads. WT RAW264.7 cells were seeded with triplicates in the 12 wells plates with 2×105 cells one day before the experiments. Cells were washed with PBS and then resuspended in lysis buffer. Cell nuclei were spined down and transferred into the transposition reaction buffer and incubated at 37 °C for 30 minutes using 105 cells. Genomic DNA was extracted using Qiagen MinElute PCR Purification Kit. ATAC-seq library was done with the protocol as previously reported (Buenrostro et al., 2015). The Purified ATAC-seq DNA library mix was sequenced on NextSeq 550 (Illumina, 35 SE reads, paired-end) in BEA Core Facility (Karolinska Institutet, Huddinge, Sweden). ATAC-Seq: The ATAC-seq Library preparation was performed through the manufacturer's introduction. 2×105 shGFP, shNCOR and shSMRT cells were seeded one day before the treatment. Total RNA was extracted from the manufacturer's introduction. RNA-Seq: The RNA-seq library preparation was followed by the instructions of the NEB Next Ultra II RNA Library kit (NEB, E7770S). 105 shGFP, shNCOR and shSMRT cells were seeded one day before the treatment. Total RNA was extracted from the manufacturer's introduction. Cut&Tag: The Cut and Tag Library preparation was performed through the manufacturer's introduction (Steven Henikoff lab,Bench top CUT&Tag V.3 ).