GSM6742508: Cocktail (chemical reprogramming)-treatment followed by ad.Atoh1 infection, (n=5), scRNA-seq; Mus musculus; RNA-Seq
Sensory epithelium from cochlear explants was dissociated to single cells using 0.25% trypsin-EDTA for 15 min incubation at 37C. Tissues were gently singularized by P1000 pipette in a complete medium (DMEM/F12 medium containing 10% FBS). Cells were strained twice through 40 mm mesh into 5 ml medium, spun down for 5 min at 300 g to pellet, and resuspended in 50 ml of complete medium. Roughly, 1400 cells of Cocktail, 1700 cells of Vehicle samples were recovered from 10X Chip-G. Single cells from cochlear cultures of Cocktail plus adAtoh1 (n=5) and Vehicle plus ad.Atoh1 (n=5) groups were captured separately to prepare NGS libraries for the samples. Single-cell suspensions were immediately transferred to individual channels of the chip (Chromium Next GEM Chip G) to generate gel beads in emulsion together with reverse transcription master mix on the droplet-based high-throughput Chromium Controller (10X Genomics) allowing cell lysis, individual cell barcoding, and reverse transcription by Chromium Next GEM Single Cell 3' Kit v3.1 (10X Genomics, PN-1000128). Briefly, single-cell library preparation was carried out by emulsion breakage, PCR amplification, cDNA fragmentation, oligo adapter, and Illumina sample index addition following the manufacturer's protocol. The single-cell derived cDNA libraries were then assessed with a bioanalyzer (High Sensitivity DNA Kit, Agilent 2100 Bioanalyzer) for quality control. Following library preparation, next-generation sequencing was performed with paired-end sequencing of 150 bp each end using Illumina HiSeqXTen (Novogene Inc.).