Zebra finch gonadal cultures DIV6, batch B, adherent fraction
The adherent cell layer was dissociated with Accutase (Sigma, Cat. #A6964) for 10min following non-adherent cell aspiration. Cells were dislodged by gentle tapping, pelleted at 250xg for 3min, and resuspended in Sorting Buffer. Trypsin was inactivated with an equal volume of PGC media before pelleting and resuspension in Sorting Buffer (Table S16). Cell suspensions were filtered through a 40m cell strainer (Bel-Art, Cat. #03-421-228) immediately before sorting on a BD FACSAria II at The Rockefeller University Flow Cytometry Resource Center (RRID:SCR_017694) for dead cell and debris exclusion using DAPI. Pelleted and FACS-sorted samples were immediately processed for scRNAseq using a 10X chromium instrument. Appropriate volumes of water and cell suspension to yield approximately 7,000 cells per sample, following the manufacturers instructions for Chromium Single Cell 3 GEM, Library & Gel Bead Kit (v3). The droplet emulsion was reverse transcribed at 53C for 45 min before heat deactivation at 85C for 5 min and cDNA amplification. cDNA was measured on a Qubit Fluorometer (ThermoFisher #Q33238) and validated for size and purity on an Agilent Fragment Analyzer (#M5310AA) using the High Sensitivity NGS Kit (Agilent, Cat. #DNF-474-0500). cDNA samples were normalized to 100ng for Illumina sequencing library preparation following the 10x Genomics protocol (Chromium Single Cell 3 Reagent Kit v3). Illumina libraries were quantified using a Qubit Fluorometer (Thermo, Cat. #Q33238) and visualized using an Agilent Fragment Analyzer (#M5310AA, DNF-474-0500). Libraries were labeled using the Chromium i7 Multiplex Kit (10X Genomics, Cat. #PN-120262) and sequenced on a NovaSeq S4 (pair-ended with read lengths of 150bp).