GSM2561495: WT_rep2; Mus musculus; RNA-Seq
RNA extraction and purification was performed using RNeasy kits (Qiagen) according to manufacturer’s instructions.  Total RNA quality is determined by estimating the A260/A280 and A260/A230 ratios by nanodrop.   RNA integrity is determined by running an Agilent Bioanalyzer gel, which measures the ratio of the ribosomal peaks. mRNA was purified from approximately 500ng of total RNA with oligo-dT beads and sheared by incubation at 94C.  Following first-strand synthesis with random primers, second strand synthesis was performed with dUTP for generating strand-specific sequencing libraries.  The cDNA library is then end-repaired, and A-tailed, adapters are ligated and second-strand digestion is performed by Uricil-DNA-Glycosylase. Indexed libraries that meet appropriate cut-offs for both are quantified by qRT-PCR using a commercially available kit (KAPA Biosystems) and insert size distribution determined with the LabChip GX or Agilent Bioanalyzer. Samples with a yield of ≥0.5 ng/ul are used for sequencing.
GEO Accession
GSM2561495