GSM8689753: IntactMouseProstate_C57BL6_ATAC; Mus musculus; ATAC-seq
Mouse prostate tissues from both intact and castrated mice were used to generate Chromium Next GEM Single Cell Multiome ATAC + Gene Expression (10x Genomics) libraries following manufacturer's protocol. Briefly, nuclei were prepared from single cells derived from tissue dissociation following the manufacturer's recommended protocol for primary cells as described in CG000365 and nuclei numbers were estimated using Countess II FL automated cell counter. Nuclei transposition were performed for ~6000 targeted nuclei recovery. Following transposition, sequential GEM (Gel Beads-in-emulsion) generation and barcoding steps enables the production of barcoded, full-length cDNA from poly-adenylated mRNA for geneexpression (GEX) library and attachment of a Spacer sequence with barcode to transposed DNA fragments for ATAC library. Post GEM lysis and cleanup, a preamplification PCR step fills gaps and generates sufficient mass for library construction. From this juncture the preamplified product was split to generate ATAC library (where P5 and P7 sequences used for Illumina bridge amplification were added) and GEX library constructions. The GEX library construction step entails enzymatic fragmentation, size selection, and addition of P5, P7, i7 and i5 sample indexes, and TruSeq Read 2 (read 2 primer sequence) via End Repair, A-tailing, Adaptor Ligation, and PCR steps. The generated libraries' size and quality were estimated using BioAnalyzer high sensitivity chip. Multiome ATAC libraries comprise double stranded DNA with standard Illumina® paired-end constructs which begin with P5 and end with P7,  Multiome GEX libraries comprise cDNA insert with standard Illumina® paired-end constructs which begin with P5 and end with P7. Sequencing was carried out on an Illumina platform, and data analysis was executed using the 10x Genomics Cell Ranger software, which provided demultiplexing, alignment, and gene quantification against the mm10 mouse genome reference.