GSM5599388: 72_RAW_WT_CRISPR_Con_H3K4me1_rp2; Mus musculus; ChIP-Seq
ChIP-Seq: Fresh cells were crosslinked with 2 mM disuccinimidyl glutarate (DSG) for 30 min, followed by 1% formaldehyde for 10 min at room temperature. Nuclei were isolated, sonicated and incubated with magnetic bead-antibody complexes.  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. ATAC-Seq: WT RAW264.7 cells were seeded with triplicates in the 12 wells plates with 2×105 cells one day before the experiments. Cells were harvested with IL4 1h treatment, 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. 4C-Seq: Fresh cells were crosslinked with 2% formaldehyde for 10 min at room temperature. Cells were counted and aliquoted with 10x7 cells per tube. For each 4C sample, 1 ml lysis buffer was applied for 10 min. Nuclei were spined down and discarded the supernatant. The nuclei were treated with the first digestion enzyme (Dpn II) overnight. The next day, the cell nuclei were heat-inactivated at 65C for 20 min and then treated with the first ligation enzyme overnight at room temperature. The genomic DNA was extracted and followed with the second digestion enzyme (Bfa I) overnight. Then heat-inactivated the enzyme and applied with then second the ligation enzyme overnight. The genomic DNA was purified again by phenol-chloroform extraction. 4C-Seq: PCR purified DNA (by baits) 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. RNA-Seq: 2×105 WT and GPS2 KO BMDM cells were seeded one day before the treatment. The next day the cells were treatment with IL4 6h and the 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). Cut&Tag: 2×105 shGFP and shKDM1A  RAW cells were seeded one day before. Cut&Tag: The Cut and Tag Library preparation was performed through the manufacturer's introduction (Steven Henikoff lab,Bench top CUT&Tag V.3 ).