Computational protocol: Hepatitis C Virus Core Protein Promotes miR-122 Destabilization by Inhibiting GLD-2

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Protocol publication

[…] Total RNA was extracted from human liver biopsies (N-1, N-2, HCV-1, HCV-2, and HCV-3) using Trizol reagent (Invitrogen). Details on the human liver biopsy specimens used in this study are given in the Supplemental Information . The small RNA fraction was enriched from total RNA extracted from human liver biopsies (N-1, N-2, HCV-1, HCV-2, and HCV-3) using a mirVana miRNA isolation kit (Ambion, Austin, TX, USA). Similarly, small RNA fraction was prepared from Huh7 cells transiently expressing HCV core protein or infected with HCV. The cDNA library for small RNA was prepared for Illumina sequencing using a Truseq Small RNA Sample Preparation kit (Illumina, San Diego, CA, USA), according to the manufacturer’s protocol. The amplified PCR products were analyzed by electrophoresis on an agarose gel, and the DNA band of an appropriate size was then excised from the gel. The cDNA amplicons were analyzed on a Bioanalyzer High Sensitivity DNA chip (Agilent, Santa Clara, CA, USA). The deep sequencing libraries were sequenced on an Illumina Genome Analyzer GA II (Illumina) using the 54-bp single read protocol. Sequencing data were analyzed using the bowtie-1.0.1 program. [...] Solexa sequence reads were subjected to adapter removal using the FASTX-toolkit (http://hannonlab.cshl.edu/fastx_toolkit/). After removal of redundant reads, the reads (16–30 nt) were mapped to the human reference genome hg19 in the UCSC Genome Browser Database [] using the Bowtie program (version 1.0.1) with the following options: -v 2 -m 10 –best—strata. The reads that could be aligned to the human genome were moved to the “mapped” dataset, and suppressed reads were discarded to prevent multiple mapping. After mapping, only unique reads of “mapped” dataset were annotated using the miRBase database (release 19) (http://www.mirbase.org) for miRNA []; the Rfam database (release 11) (http://rfam.xfam.org) for rRNA, various noncoding small RNAs, and repeat sequences []; and the genomic tRNA database (http://gtrnadb.ucsc.edu/Hsapi19/) []. Individual mapped sequence reads were compiled into a set of unique sequences with the read counts for each sequence reflecting the relative abundance. The unique sequence read counts were normalized to the total read counts of the “mapped” dataset in millions to give reads per million. […]

Pipeline specifications

Software tools Bowtie, FASTX-Toolkit
Applications sRNA-seq analysis, Genome data visualization
Organisms Hepacivirus C, Homo sapiens
Diseases Hepatitis C, Leukodystrophy, Globoid Cell
Chemicals Nucleotides