GSM8995002: mESC_SG_NT_G9a-KO_Input_spike-in_lot7; Mus musculus; ChIP-Seq
mESCs and S2 cells were cross-linked with 25 mM DSG (Thermo Fisher Scientific) for 40 min, followed by 1% formaldehyde for 15 min, and then quenched with 100 mM glycine. Cells were lysed in ChIP Lysis buffer (10 mM Tris-HCl pH 7.5, 10 mM NaCl, 0.5% NP-40, and protease inhibitor cocktail (Roche, USA)). The precipitate was suspended in SDS buffer (50 mM Tris-HCl pH8.0, 1% SDS, 10 mM EDTA) and 3x volume of 1% Triton buffer (15 mM Tris-HCl pH8.0, 150 mM NaCl, 1 mM EDTA, 1% TritonX-100 and protease inhibitor cocktail) and sonicated using Picoruptor (Diagenode). For spike-in quantification, an aliquot was reverse cross-linked in Salting Method Buffer (20 mM Tris-HCl pH8.0, 400 mM NaCl, 10 mM EDTA, 0.5% SDS) and incubated at 2 h for 60℃. DNA was purified using ChIP DNA Clean and Concentration Kit (Zymo Research, USA), and quantified using Qubit dsDNA High sensitivity Quantification Assay Kit (Thermo Fisher Scientific). mESC and S2 extracts were mixed 9:1 (DNA content) and incubated with 1 μg antibody-bound Sera-Mag SpeedBeads Protein A/G (Cytiva, USA) for 2 h at 4℃. For Lamin B1 spike-in ChIP-seq, 1 μg of anti-Lamin B1 antibody was used together with 0.5 μg of anti-H2Av antibody. Beads were washed sequentially with Low salt buffer (20 mM Tris-HCl pH 8.0, 150 mM NaCl, 2 mM EDTA, 0.1% SDS, 1% Triton X-100), High salt buffer (same, 500 mM NaCl), LiCl buffer (10 mM Tris-HCl pH 8.0, 25 mM LiCl, 1 mM EDTA, 1% deoxycholate), and TE buffer (20 mM Tris-HCl pH 8.0, 1 mM EDTA). Chromatin complexes were eluted by Proteinase K (1 mg/mL in Salting Method Buffer, 60°C, 2 h), and DNA was purified using ChIP DNA Clean and Concentration Kit. ChIP and input DNA were end-repaired, ligated to sequencing adapters, and amplified using ThruPLEX DNA-Seq Kit (TaKaRa). After size-selection and purification with AMPure XP beads (Beckman Coulter, USA), DNA libraries were sequenced with 150-nt paired-end reads on the Illumina Novaseq X Plus (illumina).