GSM7102945: VIB 10xv1 1 (v1 V1); Homo sapiens; ATAC-seq
Cryopreserved human PBMCs from a male and female donor were puchased from AllCells and (AllCells, Lot #3030456 and 3030663). Cryopreserved PBMC were thawed according to the 10x Genomics demonstrated protocol CG00039 ("Fresh Frozen Human Peripheral Blood Mononuclear Cells for Single Cell RNA Sequencing"). Briefly, 1 mL of frozen cells was rapidly thawed in a 37 °C water bath and transferred to a 50 mL tube using a 1000 µL wide bore tip. Next, 1 mL of 37 °C pre-warmed medium supplemented with 10% FBS (Thermo Fisher Scientific) was added drop-wise while gently swirling the sample. After 1 minute of incubation at room temperature, 2, 4, 8 and 16 mL of medium with 10% FBS were added drop-wise with 1 minute of incubation at room-temperature inbetween. The cell suspension was then centrifuged at 300 g for 5 min. at room temperature. The pellet was resuspended in 10 mL of medium with 10% FBS, and cells were counted. Unless specified otherwise in technology-specific methods sections, nuclei isolation was performed according to the 10x Genomics demonstrated protocol “Nuclei Isolation for Single Cell ATAC Sequencing”. Briefly, 1 million cells from the cell mix were transferred to a 1.5 mL microcentrifuge tube and centrifuged at 500 rcf for 5 min at 4 °C. The supernatant was removed without disrupting the cell pellet and 100 µL of chilled Lysis Buffer (10 mM Tris-HCl (pH 7.4); 10 mM NaCl; 3 MgCl2; 0.1% Tween-20; 0.1% Nonidet P40 Substitute; 0.01% Digitonin and 1% BSA) were added and pipette mixed 10 times. Samples were then incubated on ice for 3 min. Following lysis, 1 mL of chilled Wash Buffer (10 mM Tris-HCl (pH 7.4); 10 mM NaCl; 3 MgCl2; 0.1% Tween-20 and 1% BSA) was added and pipette mixed. Nuclei were centrifuged at 500 rcf for 5 min at 4 °C and the supernatant removed without disrupting the nuclei pellet. Based on the starting number of cells and assuming a 50% loss during the procedure, nuclei were resuspended into the appropriate volume of chilled Diluted Nuclei Buffer (10x Genomics) in order to achieve a nuclei concentration of 925-2300 nuclei/µL, suitable for a Target Nuclei Recovery of 3000. scATAC-seq libraries were prepared according to the Chromium Single Cell ATAC Reagent Kits v1.0 User Guide (10x Genomics; CG000001 Rev D). Briefly, the Transposition Reaction was prepared by mixing the desired number of nuclei with ATAC Buffer (10x Genomics) and ATAC Enzyme (10x Genomics), and was then incubated for 60 min at 37 °C. Nuclei were partitioned into Gel Bead-In-Emulsions (GEMs) by using the Chromium Controller. After this, the DNA linear amplification was performed by incubating the GEMs at the following thermal cycling conditions: 72 °C for 5 minutes, 98 °C for 30 seconds, 12 cycles of 98 °C for 10 seconds, 59 °C for 30 seconds and 72 °C for 1 minute. GEMs were broken using the Recovery Agent (10x Genomics), and the resulting DNA was purified by sequential Dynabeads and SPRIselect reagent beads clean-ups. Libraries were indexed by PCR using the Single Index Kit, and incubating at the following thermal cycling conditions: 98 °C for 45 seconds, 10 cycles of 98 °C for 20 seconds, 67 °C for 30 seconds, 72 °C for 20 seconds with a final extension of 72 °C for 1 min. Sequencing libraries were subjected to a final bead clean-up SPRIselect reagent.