alias: GSM2033167
title: GSM2033167: sRNA_LC1_a; Sus scrofa; miRNA-Seq
library_construction_protocol: Total RNA was extracted using Trizol reagent (Invitrogen, Carlsbad, CA, USA) in accordance with the manufacturer’s instructions. Total RNA was treated with RQ1 DNase (Promega, Madison, Wisconsin, USA) to remove DNA. For each sample, 10 μg of total RNA was used for RNA-seq library preparation. Polyadenylated mRNAs were purified and concentrated with oligo(dT)-conjugated magnetic beads (Invitrogen, Carlsbad, California, USA) before use for directional RNA-seq library preparation. Purified mRNAs were iron fragmented at 95 °C, followed by end repair and 5′ adaptor ligation. Reverse transcription was performed with the RT primers harboring the 3’ adaptor sequence and randomized hexamers. The cDNAs were purified and amplified. The PCR products corresponding to 200–500 bp were purified, quantified and then stored at –80 °C until used for sequencing. Total RNA (3 μg) was used for sRNA cDNA library preparation with the Balancer NGS Library Preparation Kit for sRNA (GnomeGen, San Diego, CA, USA) in accordance with the manufacture's instructions. In brief, RNAs were sequentially ligated to 3′- and 5′-end adaptors, reverse transcribed to cDNA and then amplified by PCR. Whole libraries were applied to 10% native PAGE gel electrophoresis; bands corresponding to mRNA insertion were cut and eluted. After ethanol precipitation and washing, the purified sRNA libraries were quantified with a Qubit Fluorometer (Invitrogen, Carlsbad, California, USA) before cluster generation and 36 nt single-end sequencing analysis using the Illumina GAIIx (Illumina, San Diego, CA, USA) in accordance with the manufacturer’s instructions.
identifiers.namespace: GEO
identifiers.id: GSM2033167