GSM8129337: Naive-H9_Cuttagseq_H3K9ac_rep2; Homo sapiens; OTHER
the sample used to perform RNA-seq, ATAC-seq, CUT&Tag-seq are prepared based on the commonly used method. as for CUT&Tag, 20000~50000 cells per sample replicate were washed in Wash Buffer [1 mL 1 M HEPES pH 7.5 (Sigma–Aldrich, H3375), 1.5 mL 5 M NaCl (Sigma–Aldrich, S5886-1KG), 12.5 μL 2 M Spermidine (MCE, HY-B1776), Roche Complete Protease Inhibitor EDTA-Free tablet (Sigma–Aldrich, 4693132001), and bring the final volume to 50 mL with dH2O], then immobilized on 10 ul of Concanavalin A-coated beads (Bangs Laboratories, BP531). Cells were cleared on a magnetic rack, then permeabilized with cold Antibody buffer [20 mM HEPES pH 7.5, 150 mM NaCl, 2mM EDTA, 0.1% BSA, 0.5 mM Spermidine and 1× protease inhibitor cocktail containing 0.05% Digitonin (AbMole, M5020-100mg)] on ice. The cells were then incubated with anti-H3K9me3(Abcam, ab8898, 1:100 dilution), anti-H3K9ac (Abcam, ab32129, 1:100 dilution), anti-H3K27me3(Millipore, 07-449, 1:100 dilution), H3K27ac (Abcam, ab177178, 1:100 dilution), anti-CTCF (Millipore, ab128873,1:100 dilution) for 2h at RT on a shaker. The primary antibody was cleared on a magnetic rack. Goat anti-Rabbit IgG secondary antibody (Millipore, 07-449) was diluted 1:100 in Dig-wash buffer (20 mM HEPES pH 7.5, 150 mM NaCl, 0.5 mM Spermidine and 1× protease inhibitor cocktail containing 0.05% Digitonin) and incubated at RT for an hour. Cells were cleared on a magnetic rack and washed three times with 700ul of Dig-wash buffer. A 1:100 diluted of pA-Tn5 adapter complex combining adapter primers (Table S3) and pA-Tn5 according to the manufacturer's instructions (Vazyme, S603-01) was prepared in Dig-300 Buffer [20 mM HEPES pH 7.5, 300 mM NaCl, 0.5 mM Spermidine and 1× protease inhibitor cocktail containing 0.05% Digitonin]. Cells were cleared on a magnetic rack and incubated by adding 100 ul of pA-Tn5 at RT for 1 h. Cells were washed with 700ul of Dig-300 buffer, resuspended in 300 ul of Tagmentation buffer [10 mM MgCl2 in Dig-300 Buffer], and incubated at 37 °C for 1 h. 10 ul of 0.5 M EDTA, 3 ul of 10% SDS, and 2.5 ul of 20 mg/mL Proteinase K was added to each reaction to stop the tagmentation at 37 °C overnight. DNA was purified using phenol/chloroform/isoamyl alcohol (PCI) extraction followed by chloroform extraction and precipitated with glycogen and ethanol. DNA was pelleted with a high-speed spin at 4 °C, washed, air dried for 5 min and resuspended in 25 ul of double-distilled water (ddH2O) containing 100ug/ml RNase. The DNA was then PCR amplified using TruePrep Index Kit V4 for Illumina (Vazyme, TD204), and cleaned up with VAHTS DNA Clean Beads (Vazyme, N411-01).