GSM6043370: H9, MSL3 KD ATAC Rep1; Homo sapiens; ATAC-seq
ATAC-seq is performed as described in (2) with the modifications. The freshly harvested 50,000 cells were washed in PBS and resuspended in a resuspension buffer (10mM Tris-HCl, 10mM NaCl, 3mM MgCl2). Cells were resuspended and incubated on ice for 3 min in 50ul of cold lysis buffer (0.1% NP-40, 0.1% Tween-20, 0.01% Digitonin in resuspension buffer). Nuclei were washed in 1 ml of wash buffer (990ul resuspension buffer, 0.1% Tween-20) by inverting three times. Nuclei were pelleted by centrifuging at 500 xg for 10 min at 4C.  The nuclei were resuspended in 47.5 ml of Nextera Tagmentation buffer (Nextera DNA Sample Preparation Kit) and incubated with 2.5 μl of the Tn5 transposase (Nextera kit, Illumina) at 37°C for 30 min. The resulting DNA fragments were purified using a miniElute column (Qiagen) and amplified by NEBNext High-Fidelity PCR Master Mix in a total volume of 50 μl. The thermocycling protocol for this reaction was 72°C for 5 min, 98°C for 30 s and five cycles of 98°C for 10 s, 63°C for 30 s and 72°C for 1 min. Using the universal adapter primer and a unique barcoded adapter primer (same as CUT&Tag primers). To avoid over-amplification, after the initial five cycles, the number of remaining cycles required was estimated for each sample using qPCR by adding SYBRGreen and using five μl of the previous PCR as a template. The number of additional cycles was determined to be the number it took for the qPCR to reach one-third maximal fluorescence. The original PCR was then resumed, and each sample cycled as necessary. After amplification, the samples were purified using AMPure XP beads. The libraries were sequenced as a Minimum of 50 million 150 bp paired-end reads in Novoseq (Novogene PLC).