GSM8565963: SMC Donor5, Proliferative, IgG; Homo sapiens; OTHER
Cleavage Under Targets and Release Using Nuclease (CUT&RUN) sequencing was performed using the assay kit from Cell Signaling Technology (cat# 86652). Briefly, SMCs were cultured in both quiescent and proliferative conditions, to an average of 1 million cells per plate. Cells were harvested with trypsin and washed with a wash buffer (20 mM HEPES-NaOH pH 7.5, 150 mM NaCl, 0.5 mM spermidine, and protease inhibitor cocktail). Cells were permeabilized with digitonin and incubated with activated concanavalin A magnetic beads. Cell-bead conjugates were then resuspended in 100 µL of digitonin buffer (wash buffer with 2.5% digitonin solution) containing 2 uL of Anti-Tri-Methyl-Histone H3 (Lys4) (CST, #9733), 1 uL of Anti-Acetyl-Histone H3 (Lsy 27) (CST, #8173), 2 uL of Anti-H2BK20ac (#34156), or rabbit IgG (CST, #66362), rotated overnight at 4°C, resuspended in 250 µL of antibody buffer and 7.5 µL of the pAG-MNase enzyme (#57813, CST), followed by the rotation at 4°C for 1 h. After MNase-induced cleavage, genomic DNA fragments were extracted using DNA spin columns (CST, #14209). Samples were prepared for sequencing with the DNA Library Prep Kit for Illumina (CST, #56795) combined with Multiplex Oligos for Illumina® (Dual Index Primers) (CST, #47538). The adaptor was diluted 1:125 to avoid contamination. The PCR enrichment step ran 20 cycles to amplify the adaptor-ligated CUT&RUN DNA.