GSM8285821: unselected.rat.7.CS; Rattus norvegicus; OTHER
Rat cells were isolated from the testes of 3- to 4-month-old Sprague Dawley rats transgenic for the LacZ gene under the metallothionine promoter67. Mouse cells were isolated from C57BL/6 inbred mice also 3-4 months old. Tissue was chopped into fine pieces and incubated in collagenase (Sigma) at a concentration of 1mg/ml in HBSS (Gibco) for 15 minutes at 37C. Cells were spun down for 1 minute at 600 rcf, then resuspended in warm Trypsin (Gibco, 0.25%) with 20% DNase solution (Sigma, 7mg/ml dissolved in HBSS). Tissue was pipetted for 2 minutes with a 10 ml pipette and incubated at 37C for 5 minutes. Then tissue was pipetted for another 2 minutes and incubated at 37C for 3 minutes. FBS (Sigma F2442) was added to stop the digestion. Additional DNase was added until no turbidity was visible. Cells were washed in PBS-S twice (PBS [Gibco] with 1% FBS, 10 mM HEPES [Sigma Aldrich], 1 mg/ml glucose [Sigma Aldrich], 1 mM pyruvate [Gibco], 50 units/ml penicillin [Gibco], 50 ug/ml streptomycin [Gibco] prepared as described in 68). All centrifugation steps were 5 minutes at 600 rcf.   Cells were processed both as unselected samples and enriched for EPCAM as described below. Each biological replicate was then used for either encapsulation of cells with antibody treatment or further processed as nuclei for GEX+ATAC assay. Cells were encapsulated and libraries generated for GEX+CITE using a Chromium Next GEM Single Cells 3' Kit v3.1 with Feature Barcoding (10X Genomics) per manufacturer's protocol. Prepared nuclei were encapsulated with the Chromium Next GEM Single Cell Multiome ATAC + Gene Expression kit following manufacturer's instructions. Each biological replicate was encapsulated individually. Libraries were sequenced on a NextSeq2000 sequencer (Illumina) using a 100-cycle sequencing kits to a minimum depth of 30k reads per cell.