title: GSM3460564: Moderate IOP Replicate 2; Mus musculus; RNA-Seq
library_construction_protocol: RGCs were isolated through an improved immunopanning technique.  RNA was harvested using Trizol reagent. 100ng of total RNA per mice for each treatment group were pooled and split into triplicate for the contruction of sequencing libraries. RNA sequencing libraries were generated according to SMART-seq v4 Ultra low input RNA kit (#634888, Takara Clontech, Mountain View, CA, USA). In brief, purified RNA was incubated with lysis buffer for 5 minutes, 3-SMART-seq CDS primer II (modified Oligo-dT primer) and V4 oligonucleotide were added to isolated RNA for first stranded cDNA synthesis. cDNA was amplified using PCR Primer II A, and subsequently purified using Ampure XP beads (#A63880, Beckman Coulter Life Science, Indianapolis, IN, USA). Illumina library was prepared using Nextera XT DNA library preparation kit (#FC-131-1024, Illumina, San Diego, CA, USA), and sequenced using Illumina HiSeq 2500 using 2x100bp flow cell for 20 million paired-end reads per sample