GSM2937045
GSM2937045: 2 siNFE2L2 PBS; Homo sapiens; miRNA-Seq
Total RNA was isolated by a phenol–chloroform extraction. At 48 hours after transfection, the cells were washed with PBS, scraped with 400 μL of Fenozol (A&A Biotechnology) and vigorously mixed with 100 μL of chloroform. After centrifugation (20 minutes, 10 000g, 4°C), an upper aqueous phase was collected and precipitated with 50% isopropanol overnight. RNA was pelleted by centrifugation (30 minutes, 10 000g, 4°C). After washing twice with ethanol, RNA pellet was dissolved in nuclease-free water. Small RNA fraction (with length not exceeding 50nt) was subjected to hybridization and ligation with TruSeq Small RNA Library Preparation kit (Illumina Inc.). Subsequently, RNAs were reverse transcribed and sequenced using whole-transcriptome sequencing on the Illumina NextSeq 500 platform. To detect all human miRs, a library of reference sequences was prepared by identifying the sequences of mature miRNAs within the hairpins deposited in miRBase, version 19 (Kozomara and Griffiths-Jones, 2011). The Illumina NextSeq platform requires a minimum read length of 75 nucleotides, thus all the small RNAs were extended using specific adapters that were annealed to their 3'ends during library preparation. The removal of adapters was performed in silico on the raw, 75-nucleotide- long Illumina sequence reads using cutadapt software (Martin, 2011). The resulting sequences of the length of 15-28 nucleotides were subjected to further analysis as potential miRNAs. The sequences were mapped on the prepared reference library using Bowtie v 0.12.7 (Langmead et al., 2009), with the requirement of perfect matching.
GEO
GSM2937045
GEO Accession
GSM2937045