GSM5531688: ISL1CKO_1; Mus musculus; RNA-Seq
Spiral ganglia were micro-dissected in Dulbecco's PBS on ice from E14.5 embryos of either sex from four litters; spiral ganglia from both inner ears of the individual embryo per sample. Spiral ganglia were incubated in 300 µl of lysis solution (0.05% trypsin, 0.53mM EDTA Dulbecco's PBS) in 37 ⁰C, shaking at 900 RPM for 5 min. The lysis was stopped by adding 600 µl of FACS buffer (Dulbecco's PBS, 10mM EGTA, and 2% FBS). After spinning down the samples at 800 G, 4 ⁰C for 10 min, the supernatant was removed, and cell pellets were resuspended in 500 µl of ice-cold FACS buffer. Immediately before sorting, cells were passed through a 50 µm cell sieve (CellTricsTM, Sysmex Amercica Inc.) into a sterile 5 ml polystyrene round-bottom falcon to remove clusters of cells and kept on ice. TdTomato+ neurons were sorted using a flow cytometer (BD FACSAriaTM Fusion), through a 100 µm nozzle in 20 psi, operated with BD FACSDiva™ Software. 100 sorted cells were collected into individual wells of 96-well plate containing 5 µl of lysis buffer of NEB Next single-cell low input RNA library prep kit for Illumina (#E6420 New England Biolabs). Plates were frozen immediately on dry ice and stored at −80 °C. The total time from euthanasia to cell collection was ∼3 hrs. RNAseq-libraries were prepared from 6 samples per each genotype, Neurod1Cre; Rosa26tdTOM (control) and Neurod1Cre;Isl1loxP/loxP;Rosa26tdTOM (mutant), and each sample contained 100 tdTomato+ neurons. Following the manufacturer's instructions, the NEB Next single-cell low input RNA library prep kit for Illumina was used for cDNA synthesis, amplification, and library generation at the Gene Core Facility (Institute of Biotechnology CAS, Czechia). Fragment Analyzer assessed the quality of cDNA libraries. The libraries were sequenced on an Illumina NextSeq 500 next-generation sequencer. NextSeq 500/550 High Output kit 75 cycles (# 200024906, Illumina) were processed at the Genomics and Bioinformatics Core Facility (Institute of Molecular Genetics CAS, Czechia).