title: GSM2399073: A549_24h_CAF_miRNA; Homo sapiens; miRNA-Seq
library_construction_protocol: Total RNA was extracted by using TRIzol® reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions, and RQ1 DNase (Promega, Madison, WI, USA) was used to remove contaminating genomic DNA. The quality and quantity of the purified RNA was monitored at the ratios of A260/A280 and A260/230 on SmartSpec Plus Spectrophotometer (BioRad, Philadelphia, PA, USA). RNA integrity was further verified by 1.5% agarose gel electrophoresis. For RNA-seq, total RNA was extracted from each sample, 10 mg of total RNA was used for RNA sequencing (RNA-seq) library preparation. Polyadenylated mRNAs were purified and concentrated with Magnetic Beads Oligo (dT) (Invitrogen, Carlsbad, CA, USA) before used for directional RNA-seq library preparation. Purified mRNAs were iron fragmented at 95 degrees C followed by end repair and 5' adaptor ligation. Then, reverse transcription was performed with RT primer harboring 3' adaptor sequence and randomized hexamer. The cDNAs were purified and amplified and PCR products corresponding to 200-500 bps were purified, quantified and applied to Illumina NextSeq 500 system 151 nt pair-end sequencing by ABlife, Inc. (Wuhan, China). For small RNA-seq, total RNA was extracted from each sample. 3μg of each RNA sample was used for small RNA cDNA library preparation with Balancer NGS Library Preparation Kit for small/microRNA (GnomeGen, San Diego, CA, USA) based on manufacturer’s instruction. Briefly, RNAs were ligated to 3’ and 5’ adaptor sequentially, reverse transcribed to cDNA and PCR amplified. Whole library was applied to 10% native PAGE gel electrophoresis and bands corresponding to miRNA insertion were cut and eluted. After ethanol precipitation and washing, the purified small RNA libraries were quantified with Qubit Fluorometer (Invitrogen, Carlsbad, CA, USA), used for cluster generation and applied to Illumina NextSeq 500 system 75 or 151 nt pair-end sequencing according to the manufacturer’s instructions. The small RNA libraries sequencing was perfomed in ABLife, Inc. (Wuhan, China).
identifiers.namespace: GEO
identifiers.id: GSM2399073