GSM7592753: H66_neuron_brep1; Homo sapiens; ATAC-seq
Genomic DNA. We performed ATAC-seq as previously described (Buenrostro et al.(2013) Nat Methods, PMID: 24097267) with an optimized lysis buffer to lower mitochondrial contamination (Rickner et al. (2019) J Vis Exp, PMID: 30958473).Briefly, cell membranes were lysed in RSB (10mM Tris-HCL pH 7.4, 3mM MgCl2, 10mM NaCl) with 0.1% Tween-20 freshly added. After pipetting up and down, nuclei were isolated by centrifugation at 500 rcf for 10 minutes at 4C. After discarding the supernatant, nuclei were resuspended in Tagmentation DNA (TD) Buffer with 2.5 ul Nextera enzyme (Illumina) and incubated at 37C for 30 minutes. After purification on a MinElute column (Qiagen), the purified, tagmented DNA was PCR amplified using Q5 enzyme (NEB) and barcoded primers for 10 cycles (with an initial 72C, 5 minute step for Nextera end filling). The PCR product was purified via a 1.5X SPRI cleanup (Agencourt) and checked for a characteristic nucleosome banding pattern using a TapeStation (Agilent). Samples were sequenced with paired-end 37bp reads on a NextSeq (Illumina). All 3 samples (WT, CHD8-A, CHD8-B) were processed with 2 biological replicates that were separated cultured, tagmented and analyzed.