title: GSM2450501: non-silencing sample 3; Homo sapiens; RNA-Seq
library_construction_protocol: Total RNA was isolated from each sample using an Rneasy kit according to the manufacturer's instructions. After RNA isolation, genomic DNA was removed via in-solution Dnase digest, followed by Rneasy column-based purification.  Prior to analysis, LOC550643 silencing was confirmed via RT-PCR. mRNA-Seq libraries were prepared using the mRNA Library Prep Kit according to the manufacturer's instructions (Illumina). Briefly, RNA with poly-A tail was isolated using magnetic beads conjugated to poly-T oligos. mRNA was then fragmented and reverse-transcribed into cDNA. dUTPs were incorporated, followed by second strand cDNA synthesis. dUTP-incorporated second strand was not amplified. cDNA was then end-repaired, index adapter-ligated and PCR amplified. Libraries were then quantified, pooled and sequenced at paired-end 75 base pair using the Illumina HiSeq platform at the Yale Center for Genomic Analysis Core. Raw sequencing data was demultiplexed and converted into FASTQ files using CASAVA (v1.8.2). Sequenced reads were quality tested using FASTQC and aligned to the human hg19 genome using the STAR aligner version 2.4.0k. Raw gene expression was quantified across all annotated exons and differential gene expression was carried out using the edgeR package v3.6.8. using representative triplicates to compute within-group dispersion. Differentially expressed genes were defined as having an FDR < 0.05 and a log2 fold change greater than 1.
attributes.tag: GEO Accession
attributes.value: GSM2450501