title: GSM7901970: WT, P21, VNO, replicate 1, scRNAseq; Mus musculus; RNA-Seq
library_name: GSM7901970
library_construction_protocol: 2 VNOs that were harvested at 21 days postnatal were isolated and dissociated into single-cell suspension using neural isolation enzyme/papain (NIE/Papain in Neurobasal Medium with 0.5mg/mL Collagenase A, 1.5mM L-cysteine and 100U/mL DNAse I) incubated at 37ºC. The dissociated cells were then washed with HBSS and reconstituted in cell freezing medium (90% FBS, 10% DMSO). Cells were frozen from room temperature to -80°C at a -1°C/min freeze rate. The single cell suspension was sent to the Center of Functional Genomics (CFG) , generating datasets and high-throughput single-cell gene expression profiling using the 10x Genomics Chromium Platform. Single cell library preparation and sequencing were conducted by the Center of Functional Genomics (CFG) (https://www.albany.edu/genomics/ngs.html, Albany, NY) and Singulomics Corporation (https://singulomics.com/, Bronx, NY). Cryopreserved, viable single cell suspensions were thawed, washed, resuspended in cell culture media with 0.04% BSA, and counted. Viable cell suspensions were then loaded into the Chromium Controller (10x Genomics, Pleasanton, CA) to generate gel beads-in-emulsion (GEM) with each GEM containing a single cell as well as barcoded oligonucleotides. We targeted 10,000 cells to be captured per sample for a total of 5 samples. Next, the GEMs were placed in the SimpliAmp 96-well Thermal Cycler (Thermo Fisher Scentific, Wilmington, DE) and reverse transcription was performed in each GEM (GEM-RT). After the reaction, the complementary cDNA was amplified and cleaned using Silane DynaBeads (10X Genomics, Pleasanton, CA) and the SPRIselect Reagent kit (Beckman Coulter, Indianapolis, IN). Amplified full-length cDNAs from poly-adenylated mRNA were then used to generate 3ʹ Gene Expression library (Chromium Next GEM 3' Single Cell Reagent kits v3.1, dual index), following the manufacturer's instructions (10x Genomics, Pleasanton, CA). Amplified cDNAs and the libraries were measured by Qubit dsDNA HS assay (Thermo Fisher Scientific, Wilmington, DE) and quality assessed by BioAnalyzer (Agilent Technologies, Santa Clara, CA).