GSM1246869: mES_V6.5_polyA_RNA-Seq; Mus musculus; RNA-Seq
Total RNA was purified using mirVana miRNA isolation kit (Life technologies) following the manufacturer instructions. After DNase I digestion, polyadenylated RNA and total RNA depleted of ribosomal content was used for preparation of two independent RNA-seq libraries. Polyadenylated and total RNA depleted of rRNA was fragmented with divalent cations under elevated temperature. First strand cDNA synthesis was performed with random hexamers and Superscript III reverse transcriptase. Second strand cDNA synthesis was performed using RNAseH and DNA Polymerase I. In the second-strand synthesis reaction dTTP was replaced with dUTP. Following cDNA synthesis, the double stranded products were end repaired, a single "A" base was added, and Illumina PE adaptors were ligated onto the cDNA products. The ligation products with an average size of 300 bp were purified using agarose gel electrophoresis. Following gel purification, one strand of cDNA was selectively destroyed during incubation of purified double-stranded DNA products with HK-UNG (Epicentre). The adapter ligated single-stranded cDNA was then amplified with 15 cycles of PCR and PCR products were purified using gel electrophoresis.
GEO Accession
GSM1246869