GSM8219827: HTO files for Sa_d10; Mus musculus; RNA-Seq
Single cell suspensions from skin were generated by mechanical disruption (i.e. cut into very small pieces) followed by enzymatic digestion using DNase I (0.5 mg/ml) and collagenase VI (1 mg/ml) 60 min at 37°C in 50ml RPMI. Cells were washed twice in cold PBS and resuspended in FACS buffer (PBS, 2%v/v FCS, 5mM EDTA). They were then stained with anti-CD45 antibody  and cell sorted by FACS. Cells were labeled with TotalSeq cell hashing antibodies (BioLegend) according to the cell hashing protocol (https://cite-seq.com/protocol/). After labeling, 5000 cells from each of 4 mice per group were pooled and used to generate single-cell gel beads in emulsions using the Chromium Single Cell 3′ Library & Gel Bead Kit v.3 following the manufacturer's protocols. During reverse transcription, 1 µl hash tag oligo (HTO) PCRadditive primer (0.2 µM) was added to the cDNA amplification mixture to increase the yield of HTO products. After cDNA amplification, HTO-derived cDNAs were separated from mRNA-derived cDNAs using solid phase reversible immobilization (SPRI) selection and further purified, amplified, and fragmented according to the cell hashing protocol or Chromium Single Cell 3′ Reagent Kit v3 user guide (10x Genomics), respectively. For sequencing, sample indexes were added during index PCR (12 cycles), and purified libraries were sequenced on a NextSeq 550 (Illumina).