GSM5993649: Ovaries, poolA; Mus musculus; RNA-Seq
Ovaries were collected immediately after euthanasia, cleaned from any extraneous remaining tissue (fat, oviduct, etc.) and washed in PBS 3 times. All 3 ovaries from each group were pooled together in 3mL of enzymatic dissociation solution [HBSS with 1.25mg/ml Pronase, 9.2ug/mL Elastase, 100ug/mL DNaseI, 100ug/mL Dispase, 1.5mg/Ml Collagenase A]. Ovaries were further chopped using surgical scissors in dissociation media and incubated for 30 min at 37°C in a rotatory shaker. Single cell suspensions were filtered using a 40 μm cell strainer (which removed all oocytes as well), spun down and resuspended in ACK lysis buffer for 30 sec to remove red blood cells. Cells were washed in sterile PBS (PBS; Corning, #21-031-CV) before preparing for flow sorting. Single-cell suspensions were processed using the 10x Genomics Single Cell 3' v3 RNA-seq kit by Ottawa Hospital Research Institute Stem Core Laboratories. Gene expression libraries were prepared according to the manufacturer's protocol. MULTI-seq barcode libraries were retrieved from the samples and libraries were prepared independently. Final libraries were sequenced on the NextSeq 500 platform (Illumina) to reach an approximate depth of 20,000-25,000 reads/cell. 3' scRNA-seq