GSM1940207: WT.2; Mus musculus; RNA-Seq
Animals were sacrificed by cervical dislocation and brains were rapidly excised on an ice-cold plate. Total RNA was isolated from the ventral mesencephalic area of the mouse brain using the RNAqueous Kit (Ambion) according to manufactures directions. Briefly, ventral midbrain tissues were homogenized in RNA lysis buffer and stored at -80 degrees until the extraction procedure. RNA quality was assessed via the BioAnalyzer machine (Agilent 2100). Only samples with a RIN greater than 8 were used for further processing. Total RNA was used for the messenger RNA sequencing sample preparation using the TruSeq mRNA sample preparation kit (Illumina) according to the manufacturer's protocols. Briefly, 1ug of total RNA for eacht sample was used for poly(A) RNA selection using magnetic beads coated with poly-dT, followed by thermal fragmentation. The fregmented poly(A) RNA enriched samples were subjected to cDNA synthesis using Illumina TruSeq preperation kit according to the manufacturer's protocol. Briefly, cDNA was synthesized by reverse transcriptase (Super-Script II) using poly-dT and random hexamer primers. The cDNA fragments were then blunt-ended through an end-repair reaction, followed by dA-tailing, Subsequently, specific double-stranded bar-coded adapters were ligated and library amplification for 15 cycles was performed. Pooled cDNA libraries, consisting of equal concentration bar-coded samples (wt and Ercc delta/+ samples), were sequenced in one lane on the HiSeq2000 (Illumina).
GEO
GSM1940207
GEO Accession
GSM1940207