GSM8365347: 20403, Mouse ONH 12mo B6.EDA+/+; Mus musculus; RNA-Seq
ONH sections were harvested via laser capture microdissection. RNA was extracted using Qiagen Rneasy microkit (REF#74034). RNA was verified for purity and integrity using the NanoDropOne Spectrophotometer and Agilent 2100 Bioanalyzer, respectively. Ultra-low RNA input libraries were generated using the Takara “SMART-Seq v4 Low Input RNA Kit for Sequencing” (Takara, Mountain View, CA, USA) for cDNA synthesis and the Illumina NexteraXT DNA Library Preparation (Illumina, San Diego, CA, USA) kit for cDNA dual indexing. Full length cDNA fragments are generated from 1-10ng total RNA by the Takara SMART technology. cDNA fragments are fragmented and dual indexed in a single step by taking advantage of the Nextera kit's simultaneous transposon and tagmentation step. Libraries were standardized to 2nM. Paired-end 2x150bp with 30M reads/sample sequencing was performed using standard SBS chemistry on an Illumina NovaSeq6000 sequencer. Generated raw reads were qualitied controlled, aligned, and mapped to Mus musculus using Spliced Transcripts Alignment to a Reference (STAR),58 followed by estimation of mapped pair-end reads using RNA-Seq by Expectation Maximization (RSEM).