GSM8910529: Mouse ovary, 4hr post-hCG (DEF); Mus musculus; RNA-Seq
One ovary from each mouse was pooled for single-cell isolation, with a total of three ovaries per suspension (labeled ABC or DEF). The pooled ovaries were cut into quarters using insulin syringe needles and enzymatically digested in 2 mL aMEM-Glutamax supplemented with 1 mg/mL bovine serum albumin (BSA; Sigma-Aldrich, #A3311) and 1x insulin-transferrin-selenium (Sigma-Aldrich, #1884) containing 40 µg/L liberase DH (Sigma-Aldrich, #05401089001), 0.4mg/mL collagenase IV (Sigma-Aldrich, #C5138), and 0.2 mg/mL DNAse I (Sigma-Aldrich, #9003-98-9) for 15 minutes with gently agitation at 37°C and 5% CO2 for 15 minutes. Ovaries were then mechanically digested via trituration using a 1000P wide bore tip, and the suspension was strained through a pre-wet 30 µm strainer directly into aMEM-Glutamax containing 10% FBS to quench the enzymes. Any remaining pieces of ovary were returned to the incubator in 2 mL fresh digestion media for another 15 minutes, followed by repeat mechanical digestion and straining. Once the enzymatic and mechanical digestions were complete, the cell suspension was centrifuged at 300g for 10 minutes at 4℃. The supernatant was removed, and the remaining cell pellet was resuspended in 100 µL Red Blood Cell Lysis solution (Miltenyi Biotec, #130-107-677) and incubated for 10 min at 4°C. After red blood cell removal, the suspension was centrifuged again at 300g for 10 minutes at 4°C. Following supernatant removal, the resulting pellet was resuspended in 100μL of 0.025% BSA in phosphate-buffered saline without calcium or magnesium, and transferred to lo-bind Eppendorf tubes. Cell number and viability were analyzed using Nexcelom Cellometer Auto2000 with AOPI fluorescent staining method. Sixteen thousand cells were loaded into the Chromium iX Controller (10X Genomics, PN-1000328) on a Chromium Next GEM Chip G (10X Genomics, PN-1000120), and processed to generate single-cell gel beads in the emulsion (GEM) according to the manufacturer's protocol. The cDNA and library were generated using the Chromium Next GEM Single Cell 3' Reagent Kits v3.1 (10X Genomics, PN-1000286) and Dual Index Kit TT Set A (10X Genomics, PN-1000215) according to the manufacturer's manual. Quality control for the constructed library was performed by Agilent Bioanalyzer High Sensitivity DNA kit (Agilent Technologies, 5067-4626) and Qubit DNA HS assay kit for qualitative and quantitative analysis, respectively. The multiplexed libraries were pooled and sequenced on Illumina Novaseq6000 sequencer with 100 cycle kits using the following read length: 28 bp Read1 for cell barcode and UMI, and 90 bp Read2 for transcript. The single-cell library preparation and sequencing were performed at the Northwestern University NUseq core facility with the support of an NIH Grant (1S10OD025120).