title: ATAC-seq of human iPSC derived neural sphere: sphere_neuro_d107_2242-1_hSS_rep2
design: 10,000-50,000 cells were washed in ice-cold PBS, centrifuged at 4C for 5 min at 500 rcf, washed in ice-cold Resuspension Buffer (RSB, 10 mM Tris-HCl, 10 mM NaCl, 3 mM MgCl2, pH=7.4) and centrifuged again. Cells were resuspended in 50 uL Lysis Buffer (RSB plus 0.1 % NP-40 and 0.1% Tween-20) and centrifuged at 4C for 10 min at 500 rcf. Pelleted nuclei were resuspended gently in 50 uL transposition mix (25 uL 2X Tagment DNA Buffer, 2.5 uL Tn5 Tagment DNA Enzyme 1, 0.1% Tween -20, H20) and incubated for 30 minutes at 37 C. DNA was cleaned using Qiagen MinElute columns, and PCR amplified 5 cycles in a 50 uL reaction with Illumina Nextera adaptors using NEBNext High Fidelity 2x Master Mix. A side qPCR reaction was performed using 10% of the pre-amplified PCR to determine the number of additional cycles to amplify. Final PCR products were cleaned using Qiagen MinElute columns and quantified by qPCR against a PhiX standard curve prior to pooling and sequencing to an average depth of ~10M filtered, deduplicated reads per sample on the Illumina NextSeq platform. Whole tissue samples were processed as previously described (Corces et al., 2017). Briefly, tissue samples were dounce homogenized and nuclei were isolated by gradient centrifugation prior to library preparation.