GSM6253587: Aged1-bulk; Mus musculus; RNA-Seq
Total RNA from FACS-isolated monocytes was obtained using PureLink RNA Minikit (Invitrogen). RNA quality was determined by measuring A260/A280 and A260/A230 ratios via nanodrop. RNA integrity was determined by running an Agilent Bioanalyzer gel to measure the ratio of ribosomal peaks. Samples used for library preparation had RIN values greater than 8 mRNA was purified from ~200 ng of total RNA with oligo-dT beads and sheared by incubation at 94oC in the presence of Mg2+ with the KAPA mRNA HyperPrep kit (Roche). Following first-strand synthesis with random primers, second strand synthesis and A-tailing were performed with dUTP to generate strand-specific sequencing libraries. Adapter ligation with 3' dTMP overhangs were ligated to library insert fragments. Library amplification of fragments carrying the appropriate adapter sequences at both ends was undertaken. Strands marked with dUTP were not amplified. Indexed libraries were quantified by qRT-PCR using a commercially available kit (Roche, KAPA Biosystems) and insert size distribution was determined with the Agilent Bioanalyzer. Samples with a yield of ≥ 0.5 ng/µl and a size distribution of 150-300 bp were used for sequencing.