Zebra finch gonadal cultures DIV3B, non-adherent fraction
Non-adherent cells were pelleted at 250xg for 3min and resuspended in 0.05% trypsin-EDTA at 37C for 10 minutes. 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).