GSM8449992: H820 siPool 8gy ATAC-seq A; Homo sapiens; ATAC-seq
DNA was . After irradiation, 50,000 cells were removed for ATAC-Seq library preparation, the rest for further analysis to ensure effective knockdown of lnc-RAINY. The ATAC-Seq library was prepared as follows. Cells were centrifuged at 500xg at 4C for 5 min and supernatant was removed. Pellet was resuspended in RSB Buffer (10mM Tris-HCl pH 7.4, 10mM NaCl, 3mM MgCl2, 1uL/mL each of Digitonin, Tween 20 and NP40) and incubated on ice 3 min. RSB-without (10mM Tris-HCl pH 7.4, 10mM NaCl, 3mM MgCl2, 1uL/mL Tween 20) was added and the cells were centrifuged at 500xg for 10 min. Cells were resuspended in Transposition Mix (2x TD buffer 2.5uL transposase (Illumina cat#20043196), 1uL/100uL each of Digitonin and Tween 20, water and PBS). Tubes were then incubated at 37C shaking at 1000rpm for 30 min. Reaction was cleaned using Qiagen PCR Purification Kit with 3M sodium acetate. Sample was eluted in an elution buffer and prepped for PCR Amplification. Barcoded Primers were added to each sample with 2x NEBNext Master mix (NEB cat#M0541S). Samples were PCR amplified for 12 cycles and purified using Qiagen PCR Purification Kit, eluted in water. Samples were loaded on a 2% agarose e-gel and ran 3 minutes. Each lane was cut from the gel and the DNA extracted via Qiagen gel Purification kit.