GSM8727357: HGG4M(GBM4M); Homo sapiens; ATAC-seq
Nuclei from same dissociates were used for snATAC-seq and sn-RNAseq related to this data. Briefly, 50-200 mg of tissue was dounced in 4 ml of lysis buffer (0.32 M sucrose, 5 mM calcium chloride, 0.1% Triton X-100, 0.1 mM ethylenediaminetetraacetic acid [EDTA], 3 mM magnesium acetate, and 1 mM dithiothreitol [DTT] in 1 mM Tris-HCl, pH 8.0) under RNase-free working conditions. Nuclei were isolated by sucrose gradient ultracentrifugation: a sucrose solution (1.8 M sucrose, 3 mM magnesium acetate, and 1 mM DTT in 10 mM Tris-HCl, pH 8.0) was layered under the lysate solution followed by centrifugation at 4°C for 1 hour (h) at 24,400 rotations per minute. Nuclei were resuspended in 0.04% bovine serum albumin (BSA) in Ca2+/Mg2+-free phosphate buffered saline (PBS) at 1x106 cells/mL concentration and loaded to achieve optimal recovery of 6,000 sequenced nuclei/sample with minimal doublet/triplet formation. Concentrations were verified using an automated cell counter (Countless 3, ThermoFisher). Trypan blue staining was used to assess nuclei quality and quantity prior to library preparation. Nuclei  were transposed and processed according to Chromium Single Cell ATAC kit (10X Genomics, Pleasanton, CA) using manufacturer's guidelines. An estimated 10,000 nuclei were loaded onto each channel with a targeted nuclei recovery of 6000 nuclei/sample. Fragmented DNA underwent RT and cleanup followed by amplification 10X-specific sample indexing following the manufacturer's protocol. Libraries were quantified using Tapestation (Agilent) and Qubit (ThermoFisher) analysis. Libraries were sequenced in paired end mode on a NovaSeq instrument (Illumina, San Diego, CA) targeting a depth of 50,000-100,000 reads per cell.