title: GSM5112036: SHS-mock1; Homo sapiens; miRNA-Seq
library_construction_protocol: Total RNA from mock-treated or virus-infected SHS cells was isolated using RNAzol RT (MRC, Cincinnati, OH) per the manufacturer's instructions and divided into small and large RNA fractions, and quantified using a Qubit fluorimeter-based RNA assay (Invitrogen Life Tech, Carlsbad, CA). Illumina next-generation sequencing (NGS) libraries were constructed using a TruSeq® small RNA library (Illumina, San Diego, CA). Briefly, small RNA from mock-treated SHS cells (n=2/group), or RSV- or MeV-infected SHS cells (n=3/group) was isolated at 12 hpi or 24 hpi and ligated with 3' and 5' RNA adaptors using T4 RNA ligase 2 (Epicenter Biotechnologies, Madison, WI) at 28°C for 60 min.  Ligated RNA was reverse transcribed using proprietary reverse primers and the first-strand cDNA was amplified with a combination of a proprietary forward primer and reverse primers containing unique index bar codes per the manufacturer's instructions.  Amplicons were run on a Tapestation 2200 (Agilent, Santa Clara, CA) high-sensitivity DNA chip to visualize products. Indexed samples were pooled per the manufacturer's protocol and electrophoresed on a 6% TBE-urea PAGE gel. Bands corresponding to 147bp and 157bp corresponding to adaptor-ligated mature miRNA and other non-coding RNAs were cut-out and cDNAs were precipitated. Libraries were validated on a Tapestation 2200 (Agilent) using a high-sensitivity DNA chip.  Libraries were denatured for 5 min using 5M NaOH and loaded onto a MiSeq (Agilent) for sequencing by synthesis.