GSM8440453: MV154_HIV05; Homo sapiens; RNA-Seq
RNA was extracted from sorted cells lysed in RNAzol RT (Molecular Research Center), according to the manufacturer's instructions. Briefly, 0.4 volume of sterile water was added to each lysate to allow aqueous and organic phase separation. Total RNA was then extracted from the aqueous phase by isopropanol precipitation. RNA pellets were washed with 70% ethanol and resuspended in sterile water. Extracted RNA was stored at −80°C until preparation of mRNA libraries. Messenger RNA libraries were constructed as described previously using the NEBNext Ultra RNA library preparation kit (New England Biolabs). Polyadenylated transcripts were purified with oligo-dT magnetic beads, fragmented, reverse transcribed using random hexamers and incorporated into barcoded cDNA libraries. Libraries were validated by microelectrophoresis on a tapestation 4200 (Agilent), quantified with Kapa Library Quantification Kits (Roche), pooled and clustered on an Illumina S2 flow cell. Clustered flow cells were sequenced in 2x75 base paired-end runs.