alias: GSM3528747
title: GSM3528747: Cont 1021_135; Homo sapiens; RNA-Seq
library_construction_protocol: Total RNA was extracted from the cells using a PureLink RNA Mini kit (Ambion). Cell lysates were homogenized using QIAshredder spin columns (Qiagen). Two distinct methods were used to remove genomic DNA during the RNA purification: (1) homogenized lysates were filtered through a gDNA eliminator spin columns (Qiagen) and (2) on-column DNase treatment (Qiagen). The chemistry behind silica-membrane spin column-based RNA purification necessitates ethanol concentrations ≥50% (final concentration) during precipitation and wash steps in order to obtain RNA from approximately 18 nucleotides (nt) upwards. Thus, to extract all species of RNA, including those <200 nt such as miRNA, after homogenized lysates were passed through gDNA eliminator spin columns, 100% ethanol (Sigma) was used to precipitate RNA out of solution (final ethanol concentration of 50%). To this end, we additionally used Buffer RWT (Qiagen) in place of PureLink RNA Mini Kit Wash Buffer I. Finally, RNA was eluted in the Ambion® RNA Storage Solution. The concentration and quality of RNA samples was assessed using a Bioanalyzer RNA kit (Agilent Technologies) to determine the RNA integrity number (RIN). For each sample, ≥750 ng of total RNA (RIN between 7.9 to 10) was processed for RNA-Seq. Ribosomal RNA was depleted using the Ribo-Zero rRNA Removal Kit (Epicentre) and cDNA libraries were generated with ScriptSeq mRNA-Seq Library Preparation Kit (Epicentre), which enables directional sequencing through stand-specific tagging. Paired-end sequencing was performed on indexed samples and run on the HiSeq. 2500, yielding a minimum of 50 million reads/sample. Alignment of RNA-Seq reads against the human genome (GRCh38) was performed using STAR Aligner and Htseq-count was used to count the number of overlapping reads with genes. Differential expression analysis was conducted using EdgeR.
identifiers.namespace: GEO
identifiers.id: GSM3528747