Instrument: Illumina HiSeq 2500
Strategy: RNA-Seq
Source: TRANSCRIPTOMIC
Selection: size fractionation
Layout: SINGLE
Construction protocol: Mice were euthanized by cervical dislocation. Eyes were enucleated and retinas were dissected and immediately placed in RNAlater stabilization reagent (Qiagen, Valencia, CA, USA) to preserve RNA content and integrity. Total RNA was isolated from a pool of 6 retinas per library. After removal of RNALater, Qiazol (Qiagen, Germantown, MD) was added and the tissue was disrupted using a Tissuelyser II bead mill. RNA was purified from the Qiazol homogenate using the Zymo Direct-zol (Zymo Research, Irvine, CA) protocol with on-column DNase I digestion. Libraries were made from 1 µg of total RNA utilizing the Illumina (San Diego, CA) Truseq Small RNA Sample Preparation kit, amplified with 11 rounds of PCR, and size selected for micro-RNA size inserts by gel purification. Each library was prepared to incorporate a unique index sequence which permitted multiplexing of all six libraries for sequencing. Final libraries were analyzed and quantified using the Bioanalzyer and pooled in equimolar proportions. The pooled libraries were sequenced as one 50 bp single-end run on an Illumina HiSeq 2500 instrument using a rapid-run flowcell.