title: GSM4649024: Lega_45_scATAC; Homo sapiens; ATAC-seq
library_construction_protocol: Surgically resected cortical tissue was homogenized using a glass dounce homogenizer in 2 ml of ice-cold Nuclei EZ lysis buffer (#EZ PREP NUC-101, Sigma) and was incubated on ice for 5 min. Nuclei were centrifuged at 500 × g for 5 min at 4 °C, washed with 4 ml ice-cold Nuclei EZ lysis buffer and, incubated on ice for 5 min. Nuclei were centrifuged at 500 × g for 5 min at 4 °C. After centrifugation, the nuclei were washed once in 500 μl of nuclei wash buffer consisting of 10mM Tris-HCl (pH 7.4), 10mM NaCl, 3mM MgCl2, 1% BSA and, 0.1% Tween-20. Nuclei were resuspended in 500 μl of 1X Nuclei Buffer (10X Genomics). Debris was removed with a density gradient centrifugation using the Nuclei PURE 2M Sucrose Cushion Solution and Nuclei PURE Sucrose Cushion Buffer from Nuclei PURE Prep Isolation Kit (#NUC201-1KT, Sigma Aldrich). Nuclei PURE 2M Sucrose Cushion Solution and Nuclei PURE Sucrose Cushion Buffer were first mixed in 9:1 ratio. 500 μl of the resulting sucrose buffer was added to a 2 ml Eppendorf tube. 900 μl of the sucrose buffer was added to 500 μl of isolated nuclei in NSB. 1400 μl nuclei suspension was layered to the top of sucrose buffer. This gradient was centrifuged at 13, 000 x g for 45 min at 4 °C. Nuclei pellet was resuspended and washed once in nuclei wash buffer. Nuclei concentration and integrity was determined using Ethidium Homodimer-1 (EthD-1) (#E1169, Thermo Fisher Scientific). Finally, nuclei were resuspended in 1X Nuclei Buffer at concentration of 4000 nuclei/μl for single-cell ATAC-sequencing. Droplet-based single-cell ATACseq library was prepared using the Chromium Single Cell ATAC Kit Solution v1.0 (10X Genomics) and following the Chromium Single Cell ATAC Reagent Kits User Guide: CG000168 Rev B. The library was sequenced using an Illumina NextSeq 500 at the McDermott Sequencing Core at UT Southwestern.
attributes.tag: GEO Accession
attributes.value: GSM4649024