GSM7573955: 3 months old, biol replicate1-3, HTO; Mus musculus; RNA-Seq
Ovaries were harvested from 2-3 months old and 14-17 months old mice. For the 2-3 months old age group, ovaries from 6 animals were assigned to 3 replicates (ovaries from 2 animals per replicate), while for the 14-17 months old age group, ovaries from 5 animals were assigned to 3 replicates (2 replicates with ovaries from 2 animals and 1 replicate with ovary from 1 animal). All samples were dissociated into single cell suspension in digestion buffer (1x PBS, 5% FBS, 200U collagenase IV, 200U DNaseI) using gentleMacsTM dissociator with the program 37C_m_LDK1. The suspensions were washed with 5 mL 1x PBS 5% FBS 2 nM EDTA, and passed through 100µm cell strainer to remove debris. Samples were processed using Chromium Next GEM Single Cell 3' Reagent Kits v3.1 (Dual Index) (NOTE 3) as described in the manufacturer's instructions (10X Genomics) (Haque et al., 2017). In brief, aiming for 8000 cells per library (NOTE 4), single cell suspensions with more than 70% live cells (NOTE 5) were loaded onto Chromium Controller (10X Genomics) to generate gel beads-in-emulsions (GEMs). Then co-partitioned cells were lysed, primers were released from Gel Beads, and barcoded full-length cDNA was produced and amplified. 3' Gene expression libraries were generated from cDNA by fragmentation, end repair, A-tailing, adaptor ligation and index PCR amplification. The concentration and size distribution of final libraries were assessed by Qubit™ 1X dsDNA HS Assay (Thermo Fisher Scientific, catalog # Q33231) and the Fragment Analyzer System (Agilent).