GSM8121307: reg_unc_plex-unc-rep1; Nothobranchius furzeri; RNA-Seq
Freshly collected fin tissue was dissociated with 1mg/mL collagenase type II, for 5 mins, 70μm filtered, labeled with CellPlex reagents (10x Genomics), counted, stained with and 1μg/mL DAPI, 500 nM Draq5 at 4e6 cells/mL and sorted on a 6-laser BD S6 FACSymphony with a 100-μm nozzle chilled to 4°C at all times, sorting was done on Draq5 positive DAPI negative gates, samples with post-sort viability >96% were used for library preparation and sequencing. Libraries were prepared using the Chromium Next GEM Single Cell 3' Reagent Kits v3.1 with Feature Barcode technology for Cell Multiplexing (10x Genomics) according to manufacturer's directions. Resulting cDNA, short fragment libraries, and CMO libraries were checked for quality and quantity using a 2100 Bioanalyzer (Agilent Technologies) and Qubit Fluorometer (Thermo Fisher Scientific). Multiplexed gene expression and CMO libraries were pooled, as specified by manufacturer, and sequenced to a depth necessary to achieve at least 26,000 mean reads per cell on an Illumina NovaSeq 6000 instrument utilizing RTA and instrument software versions current at the time of processing with the following paired read lengths: 28*10*10*90bp