title: GSM8543716: 903_ovary; Homo sapiens; RNA-Seq
library_construction_protocol: 125mg was used as input. The pieces were sliced into smaller pieces in a precooled petri dish kept on dry ice. The sample was transferred into a 5ml tube and lysed using a lysis buffer while constantly mixing in a cold room. The lysate was passed through a 40μm mini cell strainer and collected into a clean pre-labelled 5.0 mL Eppendorf tube. 1 mL of PBS + 1% BSA was added to the tube to further dilute the sample and then centrifuged to pellet the nuclei. The supernatant was decanted, leaving behind a small volume. The nuclei were resuspended in the remaining supernatant. An aliquot of resuspended nuclei was taken and stained with DAPI at a final DAPI concentration of 1:1000. Nuclei were counted using an Neubauer Improved haemocytometer. We used 10x Chromium Next GEM Single Cell 3ʹ Reagent Kits v3.1(10x Genomics, Pleasanton, CA) for single nuclei library preparation. The libraries were prepared using the Chromium Single Cell Controller (10x Genomics, Pleasanton, CA) using the Chromium Single Cell 3′ Library & Gel Bead v2 kit. Briefly, nuclei suspensions were diluted in nuclease-free water according to manufacturer instructions to obtain a target nuclei recovery of 10,000 nuclei. Sample encapsulation was performed using The 10x  Chromium Controller. The rest of the library preparation was carried out according to the manufacturer's instructions. Twelve cycles of cDNA amplification were performed and 13 cycles were used for sample indexing. Final libraries were pooled and sequenced on one flow cell of an Illumina NextSeq 500 (Illumina, San Diego) with a read 1: 28 cycles (cell barcode and unique molecule identifier (UMI)), i7 index: 10 cycles i5 index: 10 cycles, and read 2 90 cycles (RNA read). snRNA-seq