GSM9744888: Stage 24 gonad, female, rep1; Oryctolagus cuniculus; RNA-Seq
Pregnant New Zealand White rabbits at the stages 13-21 dpc were obtained from Charles River (France branch). Dissected gonads were dissociated into single-cells by incubating them with 0.05% Trypsin-EDTA (Gibco cat#25300-054) in addition to 1/10 portion of 5% BSA (Sigma-Aldrich cat#A7030-50G) /DPBS for 7-10 minutes at 37ºC. Dissociation was facilitated by pipetting gently every 2-3 minutes. Residual connective tissue, which appeared as whitish clumps at the end of the dissociation process, was discarded. Trypsin activity was halted by adding 2 portions of 5% BSA/DPBS. Cell suspension was passed through a 40 µm FlowMi cell strainer (Fisher Scientific cat#15342931) to remove cell clumps and debris. Filtrated cells were collected by centrifugation at 250-300g for 5 minutes at 4°C. The cell pellet was resuspended in DPBS for counting and viability check using 50% v/v trypan blue staining (Sigma Aldrich cat#T8154-20ML) under a light microscope. Only samples with ≥ 80% were used for downstream single-cell library preparation. Finally cells were fixed with 98% methanol added drop by drop and stored in -80°C freezer. For ovaries collected at 24 and 28 dpc, gonads were obtained from New Zealand rabbits bred at the SAJ rabbit facility (Jouy-en-Josas, France). The two gonads from the same fetus were pooled per stage in 500 µl of Trypsin-EDTA 0.05% at 37°C for 30 min. Dissociation was facilitated by gently pipetting every 5 minutes. Tryspin activity was stopped by adding SVF for a final 2% concentration. The cell suspension was passed through a 70 µm filter, then a 40 µm FlowMi Cell Strainer (Sigma-Aldrich cat #BAH136800040) to remove cell clumps and debris. The filtrated cells were collected by centrifugation at 300g for 10 minutes. The cell pellet was resuspended in 500 µL (24 dpc) or 1 mL (28 dpc) of DMEM/F12-SVF 2%. To determine whether the obtained cells were suitable for downstream analysis (cell viability > 80%), cell viability was evaluated using trypan blue staining with a haemocytometer r (TC20, Bio-Rad cat#1450102). The cell concentration was then adjusted to around 1000 cells/μl before loading onto the single-cell chip. For the data from gonads at the stages 13-21 dpc, fixed cells were equilibrated to 4ºC on ice for at least 5 minutes. Subsequently, the cells were pelleted at 1,000g for 5 minutes at 4℃. The cells were rehydrated and gently washed with rehydration buffer (0.04% BSA, 1mM DTT (Life Technologies cat#707265ML), 0.4U/µl RNaseIn (Life Technologies cat#AM2682), 0.2U/µl SuperaseIn (Life Technologies cat#AM2696), in DPBS), using wide-bore tips (Corning Axygen Cat#T- 205-WB-C-R-S). Cell number and quality after rehydration were assessed using 50% v/v trypan blue staining. Cell suspensions were adjusted to a final concentration of 700-1,200 cells/µl. Single-cell libraries were generated using the Chromium Next GEM Single Cell 3ʹ Kit v3.1 (10X Genomics), according to manufacturer's instructions, targeting 10,000 cells per reaction. Two libraries were prepared for each developmental stage and sex, except for female gonads at stage 15, for which only one library was prepared. Libraries were sequenced to a minimum depth of 200 million reads per library using the paired-end sequencing format: Read 1, 28 cycles; I7 index, 10 cycles; I5 index, 10 cycles; Read 2, 90 cycles.  For the data from ovaries at 24 and 28 dpc, Gel-Bead in Emulsions (GEMs) were generated using a Chromium 10X Single Cell System (10X Genomics) and cDNA libraries were prepared using the Chromium Next GEM Single Cell 5' Reagent v2 (10X Genomics). Two libraries were prepared for each developmental stage. The I2BC High-throughput Sequencing Platform (https://www.i2bc.paris-saclay.fr/sequencing/ng-sequencing/; Université Paris-Saclay, Gif-sur-Yvette, France) provided the facilities and expertise for library preparation and sequencing (Paired-end 28-64 bp, Illumina NextSeq500).