GSM2650236: Rep_3_Negative; Mus musculus; RNA-Seq
RNA was isolated with TRIzol Reagent (Life Technologies) according to manufacturer’s protocol, including the addition of 10µg RNase-free glycogen (ThermoFisher). The RNA samples were then treated with DNase (New England Biolabs) to remove any genomic DNA contamination and then cleaned up with the Purelink RNA Micro Kit (Invitrogen). The DNase-treated Total RNA (250ng) was depleted of ribosomal RNA (rRNA) using the TruSeq Stranded Total RNA kit (Illumina) and quality assessed on an Agilent 2100 Bioanalyzer to confirm that 18S and 28S rRNA peaks were depleted. The rRNA-depleted RNA was converted to dsDNA libraries by following the TruSeq Stranded Total RNA sample prep kit user guide. Briefly, the RNA was fragmented, converted to cDNA, and ligated with adaptors. The adaptor-ligated DNA was then PCR amplified using 11 cycles to generate the final libraries. The final libraries were size selected and purified using 1.0 x Ampure XP beads (Beckman Coulter) then validated by the Bioanalyzer and qPCR quantified using primers that recognize the Illumina adaptors. The libraries were then pooled at equimolar ratios, quantified using qPCR (quantification of only the adaptor-ligated libraries) and loaded onto the NextSeq 500 flow cell (Illumina) at 1.8pM final concentration.
GEO Accession
GSM2650236