GSM1443089: P35_RNA-seq_rep3; Mus musculus; RNA-Seq
Total RNA was extracted using RNeasy Micro Kit from Qiagen (Hombrechtikon, Switzerland) according to the manufacturer's protocol. Genomic DNA was removed by DNase treatment and confirmed by qPCR assay. RNA integrity was measured using an Agilent 2100 Bioanalyzer, and only samples with a RIN score >8.0 were advanced for library generation. Samples were then pooled to have at least 500 ng of RNA in triplicates for each stage. 500 ng total RNA was selected for poly A+ RNA isolation using Sera-Mag oligo(dT) beads (Thermo Scientific) and fragmented with the NEBNExt DNA library Prep Master Mix Set (New England BioLabs). Complementary DNA synthesis, end-repair, A-base addition, and ligation of the Illumina PCR adaptators were performed according to Illumina’s protocol. Libraries were then selected for 300-350 bp cDNA fragments on a 3.5% agarose gel and PCR-amplified using Phusion DNA polymerase (Finnzymes) for 15-18 cycles. PCR products were then purified on a 2% agarose gel and gel-extracted. Each library was quality controlled (product size and concentration) by an Agilent 2100 Bioanalyzer. Single-end libraries were sequenced as 60mers on a Genome Analyzer II flowcell according to Illumina’s protocol at depth of ~50-80 million single-end reads per library.
GEO Accession
GSM1443089