GSM6532941: C_SD_POOL1_scATAC_lane2; Homo sapiens; ATAC-seq
Scalp punch biopsies were rinsed with ice cold 1x PBS and then lightly diced into 1-2mm pieces with a sterile razor blade. The diced sample was then dissociated using the Miltenyi Biotec Human While Skin Dissociation Kit (Product no. 130-101-540) according to the manufacturer's directions. Briefly, samples were incubated in 0.5 mL dissociation solution containing the indicated volumes of Enzymes P, A, and D for 3 h at 37℃. Following incubation, 0.5 mL of ice cold RPMI 1640 w/ 10% FBS was added to each sample and then samples were mechanically dissociated using the gentleMACs dissociator with the 'h_skin_01' program. Following dissociation, samples were briefly centrifuged and then filtered through a 70µm cell strainer. The dissociation tube was washed with additional ice cold media and then samples were centrifuged for 10 minutes at 300 x g in a swinging bucket centrifuge. After aspirating the supernatant, samples were either resuspended in 0.1mL BamBanker freezing medium (Wako Chemicals, 302-14681) and cryopreserved at -80℃, or proceeded immediately to staining for FACS. Cells were stained with anti-CD90 PE Cy7 (BD Pharmingen, order no. 561558) for 30 min at 4℃ in FACS staining buffer (PBS with 0.5% BSA), then washed with FACS buffer. Live cells were distinguished using LIVE/DEAD Fixable Aqua Dead Cell Stain Kit (ThermoFisher, order no. L34957) according to the manufacturer's directions. For cryopreserved samples, cells were thawed at 37℃ for 3 min, resuspended in RPMI + 10% FBS and washed with FACS buffer prior to staining. Aqua negative live cells were sorted as fibroblast (CD90+) and non-fibroblast (CD90-) populations. Sorted cells were counted and the CD90+ population was reduced by half before recombining with the CD90- population for further processing by single cell ATAC-seq and/or single cell RNA-seq. Following sorting, cell suspensions were centrifuged at 300 x g for 5 minutes at 4℃ and resuspended in 1x PBS with 0.5% BSA. Samples were counted using a hemocytometer and the required volume of cells was aliquoted for generation of single-cell RNA sequencing libraries. Single-cell RNA-seq libraries were prepared using the 10x Genomics Chromium Next GEM Single Cell 3′ RNA v3.1 protocol, targeting 8,000 cells per sample. After the required number of sorted cells were aliquoted for generation of scRNA-seq libraries, the remaining sample volume was used for generation of single-cell ATAC sequencing libraries. The remaining cell volume was used to prepare nuclei according to the 10x ATAC nuclei isolation protocol for 'low cell input nuclei isolation' (CG000169, Rev B). Single cell ATAC-seq libraries were prepared using the 10x Genomics Chromium Next GEM Single Cell ATAC v1.1 protocol, targeting 6,000 cells per sample. Completed scRNA-seq libraries were sequenced on an Illumina NextSeq 550 platform with 28/8/0/91 base pair cycles. Completed scATAC-seq libraries were sequenced on an Illumina NextSeq 550 platform with 33/8/16/33 base pair cycles.