GSM7091754: AP2-Regenerated, replicate 2, scATAC; Mus musculus; ATAC-seq
Single cell tissue dissociations were performed as previously described (Lukacs et al., 2010). Briefly, in dissecting medium (DMEM (#10-027-CV, Corning) with 10% (vol/vol) FBS (#10437028, ThermoFisher), 1X glutaMAX (#35050061, ThermoFisher), and 1X penicillin-streptomycin (#15070063, ThermoFisher)) prostates were dissected away from the GU tract into individual lobes from 4-6 independent mice (N=4-6). Dorsal, lateral, and ventral prostatic lobes were pooled together, and anterior lobes were processed separately. Pooled prostatic tissue was minced and transferred to digestion solution containing 1mg/ml collagenase (#17100017, ThermoFisher Scientific). Tissue was digested at 37 °C for 2 hours with constant inversion. After digestion, tissue was spun down, resuspended in Trypsin-EDTA (#25300120, ThermoFisher), and digested for an additional 5 minutes at 37 °C. Trypsin digestions was inhibited by addition of dissecting medium supplemented with 500 U DNase I (#10104159001, Millipore-Sigma). Digested tissue was next passed through 18-G and then 20-G syringes to facilitate dissociation. Dissociated tissue was finally passed through a mesh filter with a 40 mm pore size to isolate single cells. All solutions used for tissue digestion/dissociation contained 10 mM Y-27632 dihydrochloride (#1254, Tocris) to prevent anoikis. Cell viability was greater than 80% for all downstream assays. Single cell ATAC-Seq libraries are generated using the 10X Genomics platform.  Isolated nuclei suspensions are treated with Transposase to preferentially fragment open DNA and add partial adapter sequences to the ends of the DNA fragments.  Nuclei are loaded into the Chromium Controller (10X Genomics) where they are partitioned into nanoliter-scale Gel Beads-in-emulsion with a single barcode per cell.  Libraries are completed by PCR amplification incorporate remaining Illumina adapter sequences and unique sample indexes.  The resulting libraries are evaluated on D1000 screentape using a TapeStation 4200 (Agilent Technologies), and quantitated using Kapa Biosystems qPCR quantitation kit for Illumina. They are then pooled, denatured, and diluted to 300pM with 1% PhiX control library added.  The resulting pool is then loaded into the appropriate NovaSeq Reagent cartridge and sequenced on a NovaSeq6000 following the manufacturer's recommended protocol (Illumina Inc.).