GSM9465784: Ovary_O1; Mus musculus; RNA-Seq
Ovarian tissue was dissociated using the mouse and rat adult brain dissociation kit (130-107-677; Miltenyi Biotec). The single cell suspension was then loaded on a Chromium Single Cell Controller (10X Genomics; CA). cDNA libraries were prepared using the Chromium Next GEM Single Cell 3' GEM Chip G single Cell Kit (#1000120; 10X Genomics), the Chromium Next GEM Single Cell 3' GEM, Library & Gel Bead Kit v3.1 (#1000121; 10X Genomics), the Chromium i7 Multiplex Kit (#120262; 10X Genomics) and the SPRIselect Reagent (#B23318, Beckman Coutler, Indianapolis, IN) with 12 cycles of PCR amplification to yield 150-500ng of input cDNA according to the manufacturer's instructions. Quality control was completed via the Qubit Fluorometer and the Agilent 2100 HS Bioanalyzer. Libraries were pooled in groups at equal molar concentration and sequenced on a NovaSeq S1 flowcell on the Illumina NovaSeq 6000 instrument RTA v3.4.4, recipe v1.7.0 or v1.7.5 using v1.5 chemistry with the following paired read lengths: 28 bp Read 1, 8 bp I7 Index and 91 bp Read 2. With 12,000 to 16,000 cells per sample, libraries were sequenced to a depth necessary to achieve 53,000 to 82,000 mean reads per cell - around 900 million to 1 billion reads each -1456-2508 median genes per cells.