GSM5581281: mBrain_RNA_rep2; Mus musculus; RNA-Seq
The whole brains were homogenized in Tripure isolation reagent (sigma) with a glass douncing homogenizer and further lysed by passing through a 20 gauge needle. RNA was purified by chloroform extraction, and precipitated by isopropyl alcohol. Genomic DNA was removed by RNEasy plus Mini Kit. PolyA+ RNA was isolated from 5 ug total RNA using Dynabeads Oligo(dT)25, fragmented with Mg2+ contained in the 1st strand buffer at 94°C for 15 min, and reverse transcribed using Superscript III and random hexamers to synthesize the first strand. Single strand cDNA was precipitated and dTTP was removed by phenol/chloroform/isoamyl alcohol extraction and ethanol precipitation. Second strand was synthesized with dUTP to generate strand asymmetry using DNA Pol I and the E. coli ligase, and then purified by MinElute PCR Purification Kit. Double stranded DNA was end-repaired using End-It Repair Kit, tailed with deoxyadenine using Klenow exo-, and ligated to custom adapters with T4 Rapid DNA Ligase (Enzymatics). Fragments of 200-600 bp were size-selected using Agencourt AMPure XP beads (0.5X and 0.3X), and subjected to PCR amplification using Q5 DNA polymerase. Libraries were size-selected using Agencourt AMPure XP beads (0.75X), quantified by Qubit™ dsDNA HS Assay Kit and quality checked by High Sensitivity D1000 ScreenTape. Libraries were sequenced as 100 bp paired-end reads on the Illumina HiSeq 4000 platform.