Chromium next gem single cell atac reagent kits v1

For scATAC-seq library construction, the samples were cryo-preserved with CryostorCS10 (STEMCELL). The nuclei were extracted according to the 10x Genomics user guide. Cryopreserved samples were recovered in prewarmed media (RPMI 1640 + 10% FBS) and then subjected to centrifugation at 300×g for 5 min at 4 °C. After DPBS washing, the cells were lysed in chilled lysis buffer (10 mM pH 7.4 Tris-HCl, 10 mM NaCl, 3 mM MgCl₂, 0.1% Tween-20, 0.1% Nonidet P40 Substitute, 0.01% digitonin, 1% BSA) with gentle pipette mixing and incubation on ice for 3 min. After washing with chilled wash buffer (10 mM pH 7.4 Tris-HCl, 10 mM NaCl, 3 mM MgCl₂, 0.1% Tween-20, and 1% BSA), the nuclei were resuspended in chilled diluted nuclei buffer (10x Genomics) and filtered through 40-μm strainers. The quality and quantity of nuclei were manually examined by trypan blue staining. Nuclei suspensions with a target recovery of 3000-6000 nuclei were subjected to Chromium Next GEM Single Cell ATAC Reagent Kits v1.1 (10x Genomics) according to the manufacturer’s instructions. The library was processed on the Illumina NovaSeq6000 platform for sequencing with 50 bp paired-end reads.

Cryopreserved PBMCs isolated from three healthy unrelated donors were obtained from Hemacare (Los Angeles, CA, USA). PBMCs were thawed and cultured in RPMI 1640 with GlutaMax and HEPES (ThermoFisher) supplemented with 10% FBS and 1% pen/strep overnight on ultra-low-attachment 10cm dishes (Corning). Following overnight culture, suspension PBMCs were transferred to a 50mL conical tube and placed on ice. Adherent PBMCs were lifted using TrypLE and pooled with the suspension PBMCs. The cells were then washed twice with ice-cold PBS before nuclei isolation according to the “Nuclei Isolation for Single Cell ATAC Sequencing” protocol from 10x Genomics. Isolated nuclei were then resuspended in ice-cold 10x Genomics diluted nuclei buffer and counted. 15,000 nuclei from each PBMC donor were then subjected to transposition according to the “Chromium Next GEM Single Cell ATAC Reagent Kits v1.1” protocol from 10x Genomics. After transposition, nuclei from each donor were pooled, pelleted, resuspended in 10x Genomics ATAC Buffer B, and counted prior to microfluidic capture (targeting ~10,000 recovered nuclei). Next-generation sequencing library preparation was then performed according to supplier recommendations before sequencing on a single lane of an Illumina NovaSeq SP100 flow cell.

Cryopreserved PBMCs were thawed at 37°C and transferred into 50 mL centrifuge tubes. PBMCs were washed in PBS through centrifugation at 400G for 5 minutes at 4 °C and lysed for 3 minutes on ice. After discarding the supernatant, lysed cells were diluted within 1× diluted nuclei buffer (10X Genomics) prior to counting with trypan blue using a Countess II FL Automated Cell Counter to validate lysis. An equal number of nuclei (approximately 3000 nuclei per sample) from four individuals were pooled together and then loaded into the Chromium Next GEM Chip H based on the user guides from 10X genomics (Chromium Next GEM Single Cell ATAC Reagent Kits v1.1 User Guide, CG000209 Rev D). After breaking the emulsion, the barcoded tagmented DNA was purified and amplified for sample indexing and generation of scATAC-seq libraries. The final libraries were quantified using the Agilent Bioanalyzer High Sensitivity DNA kit. Sequencing was performed on NovaSeq 6000 with a depth of 25,000 reads per nuclei.