GSM9414905: mESC_TT2_H3K9me3_lot3; Mus musculus; ChIP-Seq
mESCs were cross-linked with 25 mM DSG (Thermo Fisher Scientific) for 40 min at RT, followed by 1% formaldehyde for 15 min at RT, and then quenched with 100 mM glycine. Cells were lysed with 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), then sonicated using Picoruptor (Diagenode, USA). To quantify the amount of DNA for spike-in ChIP, a portion of the sonicated cell extract was mixed with 2x volume of 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 DNA concentration was determined using Qubit dsDNA High sensitivity Quantification Assay Kit (Thermo Fisher Scientific). Extracts from mESCs and S2 cells were mixed at a ratio of 9:1 (DNA content) and incubated for 2 h at 4℃ with antibodies that were pre-incubated with Sera-Mag SpeedBeads Protein A/G Magnetic Particles (Cytiva, USA). The samples were washed consecutively with Low buffer (20 mM Tris-HCl pH8.0, 150 mM NaCl, 2 mM EDTA, 0.1% SDS, 1% Triton-X100), High buffer (20 mM Tris-HCl pH8.0, 500 mM NaCl, 2 mM EDTA, 0.1% SDS, 1% Triton-X100), LiCl buffer (10 mM Tris-HCl pH8.0, 25 mM LiCl, 1mM EDTA, 1% Deoxycolate), TE buffer (20 mM Tris-HCl pH8.0, 1 mM EDTA). The chromatin-immune complex was treated with 1mg/mL Proteinase K in Salting Method Buffer for 2 h at 60℃. 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).