GSM8457620: MORC2 WT HEK293T cell line, Replicate 2; Homo sapiens; ATAC-seq
50,000 cells were pelleted at 500 x g for 5 minutes at 4C then lysed on ice for 3 minutes in 50 μL ATAC-RSB Buffer (10mM Tris-HCl pH 7.4, 10mM NaCl, 3mM MgCl2) also containing 0.1% (v/v) IGEPAL CA-630, 0.1% (v/v) Tween-20, 0.01% (w/v) Digitonin. After lysis, 1mL of ATAC-RSB containing only 0.1% (v/v) Tween-20 but no IGEPAL CA-630 or Digitonin was added, and nuclei were pelleted at 500 x g for 10 minutes at 4C. All supernatant was aspirated and discarded, and each pellet of nuclei was resuspended in 50 μL of transposition mix (25 μL 2x TD Buffer, 2.5 μL Tn5 transposase (100 nM final), 16.5 μL PBS, 0.1 μL 5% digitonin, 0.5 μL 10% Tween-20, 5 μL H2O) and incubated at 37 °C for 30 minutes exactly in a thermomixer with 1000 RPM mixing. DNA was then purified using Zymo DNA Clean & Concentrator-5 Kit to generate 20 μL of product which was pre-amplified for 5 cycles using NEBNext 2x Master Mix (#E7649A) and indexed primers as described previously57. A 5 μL of pre-amplified DNA was then used in a 15 μL qPCR reaction using Promega GoTaq qPCR Master Mix to determine the required number of additional cycles to yield minimally amplified DNA for optimal library diversity for next-generation sequencing (determined to be 3-4 additional cycles for these libraries). Amplified DNA was then purified using NucleoMag NGS Clean-up and Size Select beads (Macherey-Nagel) using a 0.5x ratio of beads to DNA to initially clear large DNA fragments from the library and then a 1.5x ratio of beads to supernatant to adsorb DNA fragments of appropriate size for sequencing. DNA-bound beads were then washed twice with 80% EtOH and size-selected DNA was eluted in nuclease-free water. Libraries were sequenced on an Illumina NextSeq 2000 to produce 65bp paired-end reads.