title: GSM8000369: postnatal 56 days, female replicate 2, scRNAseq; Mus musculus; RNA-Seq
library_construction_protocol: Mice VNOs were dissected in cold oxygenated ACSF following Ma et al, 2011. Dissected epitheliums were dissociated in papain solution (20mg/mL papain and 3mg/mL L-cysteine in HBSS) with RNase-free DNase I (10unit) at 37°C for 15-20 minutes. 0.01% BSA in PBS was added to the digestion solution before filtering with 70µm and 30µm filters (pluriSelect). Dissociated cells were washed twice in 0.01% BSA with final volume 1mL, followed by Draq5 (25µM) and DAPI (0.5µg/mL) staining 5min on ice. Draq5+/DAPI- cells (live/nucleated cells) were sorted on BD Influx cytometer (BD Bioscience) with 100µm nozzle. Dissociated sorted cells were assessed for concentration and viability via Luna-FL cell counter (Logos Biosystems). Cells deemed to be at least 90% viable were loaded on a Chromium Single Cell Controller (10x Genomics), based on live cell concentration. Libraries were 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 Fluorometer (Thermo Fisher Scientific). With cells captured estimated at ~5,500-8,000 cells per sample, libraries were pooled and sequenced to a depth necessary to achieve at least 40,000 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*8*91bp.