Next-Generation Sequencing of Small RNAs from HIV-Infected Cells Identifies Phased microRNA Expression Patterns and Candidate Novel microRNAs Differentially Expressed upon Infection

Chang, Stewart T.; Thomas, Matthew J.; Sova, Pavel; Green, Richard R.; Palermo, Robert E.; Katze, Michael G.

Identification of candidate novel microRNAs. Candidate novel mi-croRNAs were identified by analyzing genome-mapped small RNA-Seq reads from HIV-and mock-infected samples at 5, 12, and 24 hpi using miRDeep with default parameters (15) . Candidate novel microRNAs were then filtered to include only those present with Õ†10 reads in Õ†11 out of 17 samples (i.e.,~two-thirds of samples). DE candidate microRNAs were identified using DESeq. As a final filter, we considered only candidates with nonzero expression in mock-and HIV-infected samples at the DE time point for further analysis. The UCSC Genome Browser (44) was used to access other small RNA-Seq data from the ENCODE project (16) as well as evolutionary conservation data via Multiz (45) and GERP scores (46) . qPCR. We performed qPCR for each microRNA using a miRCURY LNA universal RT microRNA PCR system (Exiqon, Woburn, MA). For each assay, we utilized 20 ng of total RNA in an ABI 7900 real-time PCR system with SYBR green-based detection (Applied Biosystems, Foster City, CA). Each assay was run in triplicate with SYBR green PCR Master Mix (Exiqon, Woburn, MA) for microRNA detection in a 10-l reaction volume.