title: GSM8372598: scATAC-seq of ccRCC for RCC76; Homo sapiens; ATAC-seq
library_construction_protocol: Suitable nuclei concentrations were adjusted from the above experiment. As mentioned in the previous study (avoiding potential cell duplets), the number of target nucleus captured in each sample was 7,000. We adopted 10x Genomics 'Chromium Single Cell ATAC Reagent Kits User Guide' for library preparation (https://support.10xgenomics.com/single-cell-atac/library-prep/doc/technical-note-chromium-next-gem-single-cell-atac-v11-reagent-workflow-and-software-updates). Briefly, the ATAC Buffer (10x Genomics, 200122) and ATAC Enzyme (10x Genomics, 200123) were added to the 5 µL nuclei, which was incubated for 60 min at 37 °C. We prepared 'Master Mix', including 56.5 µL of Barcoding Reagent B (10x Genomics, 2000194), 1.5 µL of Reducing Agent B (10x Genomics, 2000087) and 2 µL of Barcoding Enzyme (10x Genomics, 2000125). After mixing the nuclei with the Master Mix, we added it to the Chromium Chip E (10x Genomics, 2000121). At the same time, we added Partitioning Oil (10x Genomics, 220088) and Chromium Single Cell ATAC Gel Beads (10x Genomics, 2000132) to the Chromium Chip E. The Chromium Chip E was placed into a Chromium Single Cell Controller instrument (10x Genomics) before attaching a 10x Gasket (10x Genomics, 3000072). Then, the Chromium Single Cell Controller Instrument could be run. The gel bead in emulsions (GEMs) were generated after the above steps. Subsequently, GEMs were incubated and cleaned by using Dynabeads (10x Genomics, 2000048) and SPRIselect (Beckman Coulter, B23318). scATAC-seq library construction eliminates the process of reverse transcription compared with scRNA-seq. PCR was performed after 'Sample Index PCR Mix' and Single Index Kit N Set A (10x Genomics, 1000212) were added into the samples. The details of PCR parameters could be referred to Chromium Single Cell ATAC Reagent Kits User Guide. DNA libraries were sequenced by NovaSeq 6000 (Illumina, San Diego, CA). Consistent with previous studies, we followed the 2 × 50 paired-end sequencing scheme: 50 bp read 1N, 8 bp i7 index, 16 bp i5 index and 50 bp read 2N. 10X Genomics Chromium Single Cell ATAC Reagent Kits V1