Next-Generation Sequencing Reveals HIV-1-Mediated Suppression of T Cell Activation and RNA Processing and Regulation of Noncoding RNA Expression in a CD4(+) T Cell Line

Chang, Stewart T.; Sova, Pavel; Peng, Xinxia; Weiss, Jeffrey; Law, G. Lynn; Palermo, Robert E.; Katze, Michael G.

RNA preparation and next-generation sequencing. Total RNA was extracted from 5 Ï« 10 6 cells per sample using a mirVana kit (Applied Biosystems/Ambion, Austin, TX), and the quality and concentration of the RNA were determined by an Agilent 2100 Bioanalyzer. Samples were submitted for sequencing to IlluminaFastTrack Sequencing Services (Hayward, CA). cDNA libraries were generated using Illumina mRNA-SEQ kit. The quality and concentration of these libraries were determined by an Agilent 2100 Bioanalyzer. The libraries were clonally amplified on a cluster generation station using Illumina version 4 cluster generation reagents to achieve a target density of approximately 300,000 (300K)/mm 2 in a single channel of a flow cell. The resulting libraries were then sequenced on a Genome Analyzer IIx using Illumina version 4.0 sequencing reagents which generated single reads of 75 nucleotides (nt). Image analysis, base calling, and error estimation were performed using Illumina Analysis Pipeline (version 2.6).