GSM8766530: Outer hair cells, P0, WT, rep4; Mus musculus; RNA-Seq
Atoh1-GFP positive inner hair cells (IHCs) and Atoh1-GFP; Insm1-GFP-Cre and R26-TdTomato positive sensory outer hair cells (OHCs) were FACS-sorted from P0 cochleae into 350μL RNA lysis buffer (Qiagen), and RNA was extracted using RNeasy Micro Kit (Qiagen). Libraries were synthesized by the Beijing Genomics Insititute (BGI) at their facility and the Children's Hospital of Philiadelphia. Genomic DNA was first removed using DNAse I, and ribosomal RNA (rRNA) was removed using RiboZero (Epicentre).The mRNA was fragmented into short fragments of about 200bp. First strand cDNA synthesis was done using random hexamers and the NuGEN Ovation RNA-Amplification System V2. Buffer, dNTPs, RNase H, and DNA polymerase I were added to synthesize the second strand. Double-stranded cDNA was purified with magnetic beads, end reparation and 3′-end single nucleotide A (adenine) addition were performed, and sequencing adaptors were ligated to the fragments, which were enriched by PCR amplification. Libraries were qualified and quantified with an Agilent 2100 Bioanalyzer and ABI StepOnePlus Real-Time PCR. Libraries were sequenced using the Illumina HiSeq4000 sequencer using 100bp pair-end reads, to a depth of about 100 million reads per sample.