GSM4274297: cocaine-saline pooled_Iso-Seq; Mus musculus; RNA-Seq
Nac were isolated one hour after the final injection and stored frozen at -80 until RNA extraction. RNA from individual mice was extracted using Qiagen RNeasy Mini kit and pooled prior to library preparation. NAc were isolated from each mouse bilaterally, so a total of 20 NAc punches were used for the experiment. Long read isoform sequencing was performed according to the recommendations made in the Procedure and Checklist for Iso-Seq from Pacific Biosciences. We have applied Isoform Sequencing (IsoSeq) methods to the pooled sample. Briefly, two micrograms of high quality RNA (RIN = 8.8) was used as input into oligo-dT primed cDNA synthesis (Clontech). Following double-stranded cDNA amplification, transcripts were size selected, separating transcripts < 4 kb, from those > 4kb, using magnetic beads. The transcripts of each size bin were then equimolar pooled in order to adequately represent longer transcripts in the subsequent sequencing library. The pooled cDNA was then used as input into SMRTbell library preparation as recommended by the manufacturer. The SMRTbell library was sequenced on a SMRTcell 8M on the Sequel 2 platform using v2 chemistry, producing over 300 Gb of data.
GEO Accession
GSM4274297