title: GSM6436449: 10x scATAC-seq on mix of MM050, MM099, MM116, MM001, MM011, MM057 and MM087; Homo sapiens; ATAC-seq
library_construction_protocol: Nuclei were extracted from the mm lines as follows; Cells were washed with phosphate buffered saline (1x PBS; Thermo Fisher Scienific), detached using trypsin (Thermo Fisher Scientific) and centrifuged at 1,000 r.p.m. for 5 min to remove the medium after this protocol CG000169 (10x Genomics) was followed. Briefly, cells were washed with PBS + 0.04% BSA and cell concentration was determined with the LUNA-FL Dual Fluorescence Cell Counter. For each cell line, 500k cells were resuspended in 100 µl nuclei lysis buffer (10 mM Tris-HCl pH 7.4; 10 mM NaCl; 3 mM MgCl2; 0.1% Tween-20; 0.1% NP40; 0.01% Digitonin and 1% BSA in nuclease-free water). After 5 min incubation on ice, 1 ml chilled wash buffer was added to the lysed cells (10 mM Tris-HCl pH 7.4; 10 mM NaCl; 3 mM MgCl2; 0.1% Tween-20 and 1% BSA in nuclease-free water). The lysed cell suspension was centrifuged at 500 rcf for 5 min at 4°C and the pellet was resuspended in 1x nuclei buffer. Single-cell libraries were generated using the GemCode Single-Cell Instrument and Single Cell ATAC Library & Gel Bead Kit v1-v1.1 and ChIP Kit (10x Genomics). Briefly, single nuclei suspended in 1x nuclei buffer were incubated for 60 min at 37°C with a transposase that fragments the DNA in open regions of the chromatin and adds adapter sequences to the ends of the DNA fragments. After generation of nanoliter-scale gel-bead-in-emulsions (GEMs), GEMs were incubated in a C1000 Touch Thermal Cycler (Bio-Rad) under the following program: 72°C for 5 min; 98°C for 30 s; 12 cycles of 98°C for 10 s, 59°C for 30 s, 72°C for 1 min; and hold at 15°C. After incubation, single-cell droplets were broken, and the single-strand DNA was isolated and cleaned using Cleanup Mix containing Silane Dynabeads. Illumina P7 sequence and a sample index were added to the single-strand DNA during library construction via PCR: 98°C for 45s; 9-13 cycles of 98°C for 20 s, 67°C for 30 s, 72°C for 20 s; 72°C for 1 min; and hold at 15°C. The sequencing-ready library was cleaned up with SPRIselect beads.