GSM7102980: CNA mtscatac 1 (mtscATAC C1); 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. Cells from each donor were subsequently pooled at a 1:1 ratio and after washing, cells were fixed in 1% formaldehyde (FA; ThermoFisher #28906) in PBS for 10 min at RT, quenched with glycine solution to a final concentration of 0.125 M before washing cells twice in PBS via centrifugation at 400 g, 5 min, 4 °C. Cells were subsequently treated with lysis buffer (10 mM Tris-HCL pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.1% NP-40, 1% BSA) for 3 min on ice, followed by adding 1 mL of chilled wash buffer and inversion (10 mM Tris-HCL pH 7.4, 10 mM NaCl, 3 mM MgCl2, 1% BSA) before centrifugation at 500 x g, 5 min, 4 °C. mtscATAC-seq libraries were generated using the Chromium Next GEM Single Cell ATAC Library & Gel Bead Kit (v1.1, #1000175) according to the manufacturer's instructions (CG000209). Briefly, following tagmentation, cells were loaded on a Chromium controller Single-Cell Instrument to generate single-cell Gel Bead-In-Emulsions (GEMs) followed by linear PCR as described in the protocol using a C1000 Touch Thermal cycler with 96-Deep Well Reaction Module (Bio-Rad). After breaking the GEMs, the barcoded tagmented DNA was purified and further amplified to enable sample indexing (11 cycles of PCR) and enrichment of mtscATAC-seq libraries. The final libraries were quantified using a Qubit dsDNA HS Assay kit (Invitrogen) and a High Sensitivity DNA chip run on a Bioanalyzer 2100 system (Agilent).