title: GSM8653150: Pachón ovary 1; Astyanax mexicanus; RNA-Seq
library_construction_protocol: The digestive enzyme mixture is composed of 3 mg/ml collagenase I (C0130, Sigma-Aldrich), 3 mg/ml collagenase II (C6885, Sigma-Aldrich) and 1.6 mg/ml hyaluronidase (H4272, Sigma-Aldrich) in L15 media (112-029-101, Quality Biological). Fish were fasted one day before cell dissociation. Each sample and library were collected and constructed from a single fish, and the fish being used were aged-matched.  The left side ovary from one adult fish was dissected and stored on ice in an RNA Lobind tube (0030108310, Eppendorf north america) containing 3 ml of L15 for no more than 40 min. Tissues were minced with dissection scissors into pieces. L15 media were replaced with 3 ml of digestive enzyme mixture after the tissue settled down to the bottom of the tube. Samples were incubated on an orbitron rotator (260200, Boekel Scientific) at room temperature until no obvious cell clumps were observed (35~45 min, depending on samples). Cells were centrifuged at 300 g for 3 min at 4 °C, resuspended in 3 ml of TrypLE (12604021, ThermoFisher Scientific) and incubated on the orbitron rotator at room temperature for 15 min. 3 ml of 20% FBS (SH30071.03E, Cytiva) in L15 were added to the cell suspensions and incubated on the orbitron rotator at room temperature for 1 min to stop TrypLE reaction. Cell suspensions were then added with 15 ml of L15 and centrifuged at 300 g for 3 min at 4 °C to get the cell pellets. The cell pellets were resuspended and washed three times with 5 ml of L15 using a P1000 pipette and centrifuged at 300 g for 3 min. Cell pellets were resuspended in 2 ml L15 and filtered through a 70 µm nylon filter (352350, Corning) and then through a 40 µm nylon filter (352340, Corning) into Lobind tubes to remove large cells and cell clumps. The filters were washed twice with 2 ml L15. The filtrates were centrifuged at 300 g for 3 min and resuspended in 1 ml of 2% BSA (A9418, Sigma-Aldrich) in phosphate-buffered saline (PBS) (20012050, ThermoFisher Scientific) and transferred into Lobind tubes. scRNA-seq libraries were prepared by Stowers Sequencing core. Dissociated, sorted cells were assessed for concentration and viability using a Luna-FL cell counter (Logos Biosystems). Cells deemed to be at least 84% viable were loaded based on live cell concentration onto a Chromium iX Series instrument (10x Genomics) and libraries prepared using the Chromium Next GEM Single Cell 3' Reagent Kits v3.1 (10x Genomics) according to manufacturer's directions. Resulting cDNA and short fragment libraries were checked for quality and quantity using a 2100 Bioanalyzer (Agilent Technologies) and Qubit 2.0 Fluorometer (Thermo Fisher Scientific). With cells captured estimated at ~1,100-5,500 cells per sample, libraries were pooled and sequenced to a depth necessary to achieve at least 38,000 mean reads per cell on an Illumina NextSeq 2000 instrument utilizing RTA and instrument software versions current at the time of processing with the following paired read lengths 28*10*10*90bp.