GSM1196059: mmu pl I 1 smallRNA [batch2]; Mus musculus; RNA-Seq
Total RNA was extracted using the Trizol (Invitrogen) procedure or RNAeasy/RNAeasy Lipid/miRNeasy (Qiagen) column purification kits. RNA quality was assessed using an Agilent 2100 Bioanalyzer. Non-strand-specific sequencing libraries were prepared using the mRNA-Seq Sample Prep Kit (Illumina) according to the manufacturer's instructions. Briefly, polyadenylated RNA was isolated using a poly-dT bead procedure and then chemically fragmented and randomly primed for reverse transcription. After second-strand synthesis, the ends of the double-stranded cDNA were repaired. After 3'-end adenylation of these products, Illumina Paired-End Sequencing adapters were ligated to the blunt ends of the cDNA fragments. Ligated products were run on gels; 250-300 bp fragments were excised and then PCR-amplified (15 cycles). After column-purification, qualities of the resulting libraries were assessed using Agilent 2100 Bioanalyzers. Strand-specific libraries were prepared using an optimized version of the original Directional mRNA-seq Library Prep Pre-Release Protocol from Illumina. We used reagents from various companies instead of Illumina’s to decrease significantly the library cost and as a consequence adapted the reaction parameters for each step. In addition, we integrated a size selection step, right after fragmentation to optimize the insert size distribution and decrease the adapter contamination. This optimized protocol is available upon request.
GEO Accession
GSM1196059