GSM9701627: HEK293 sorbitol replicate 2 ATAC-seq; Homo sapiens; ATAC-seq
Cells were detached, counted, and pelleted at 4 °C. For RNA-seq, total RNA was extracted using a column-based RNA purification kit following the manufacturer's instructions. For CUT&Tag and Hi-C, nuclei were isolated by gentle lysis in detergent-containing buffer prior to downstream processing. For ATAC-seq, 50,000 cells per sample were used. Nuclei were isolated using Omni-ATAC lysis buffer (10 mM Tris-HCl pH 7.4, 10 mM NaCl, 3 mM MgCl₂, 0.1% NP-40, 0.1% Tween-20, 0.01% digitonin) on ice for 3 minutes, followed by a wash with 10 mM Tris-HCl pH 7.4, 10 mM NaCl, 3 mM MgCl₂, 0.1% Tween-20. Isolated nuclei were used immediately for tagmentation. ATAC-seq libraries were prepared following the Omni-ATAC protocol (Corces et al., 2017). 50,000 cells per sample were lysed in ATAC lysis buffer (10 mM Tris-HCl pH 7.4, 10 mM NaCl, 3 mM MgCl₂, 0.1% NP-40, 0.1% Tween-20, 0.01% digitonin) on ice for 3 minutes. Nuclei were pelleted, washed once with wash buffer (10 mM Tris-HCl pH 7.4, 10 mM NaCl, 3 mM MgCl₂, 0.1% Tween-20), and resuspended in transposition mix containing Nextera XT transposase (Illumina) for 30 minutes at 37 °C with gentle shaking. Transposed DNA was purified using the Zymo DNA Clean and Concentrator kit. Libraries were amplified by PCR for 5 initial cycles; a qPCR side reaction on 5% of the amplified material was used to determine the number of additional cycles required (typically 4–7 additional cycles). Libraries were size-selected using a two-sided AMPure XP bead cleanup (0.5× then 1.3×), quantified by Qubit (dsDNA HS Assay) and the KAPA Library Quantification kit, and sequenced in paired-end 75 bp mode on an Illumina NextSeq 500.