alias: GSM2891092
title: GSM2891092: siSRSF1-3; Homo sapiens; RNA-Seq
library_construction_protocol: Total RNA was isolated from viable cells, 48 hours after siRNA transfection with siSF3B1 and the siNon-Targeting (siNT) control, and 72 hours after transfection with siSRSF1 and its siNT control using Trizol reagent (15596; Invitrogen, Breda, The Netherlands) and the miRNeasy Mini Kit (217004; Qiagen, Venlo, the Netherlands), following the manufacturer's protocol. Concentrations and purities were measured on a Nanodrop ND-1000 spectrophotometer (Isogen, Ijsselstein, The Netherlands). cDNA libraries were prepared with the TruSeq Stranded mRNA LT sample Prep kit (RS-122-2101, Illumina, San Diego, USA) according to the TruSeq Stranded mRNA sample preparation guide (Part# 15031047, Revision E, October 2013). cDNA library quality control was performed using the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, USA). Sample libraries were diluted and pooled to obtain a final concentration of 10 nM. Sequencing was performed on an Illumina HiSeq V4 2500, using a 125 bases paired end run with an input of 16 pM cDNA RNA isolated from siSF3B1- and siNT-treated SW480 cells was subjected to full-length RNA single molecule real time (SMRT) sequencing called Iso-Seq(34). Briefly, RNA (RIN score of ~9.0 assessed by Agilent Bioanalysis) was amplified using the ClonTech Switching Mechanism at 5' end of RNA Template (SMART) technology which incorporates known sequence at both ends of the cDNA product in the first strand synthesis process without the need for conventional adapter ligation strategies. 408 ng of siSF3B1 and 352ng siNT cDNA were used as input to the SMART cDNA amplification process to capture full-length, intact isoforms to be reverse transcribed and amplified into full-length cDNA representing the full transcriptome where the known sequences are used to complete SMRTbell library preparation using the cDNA products. Once ample double stranded cDNA was synthesized, cDNA Iso-Seq sequencing libraries were prepared using the SMRTbell library preparation procedure resulting in a library containing molecular inserts that represent a single isoform per library molecule. These libraries were then size-selected to enrich for isoforms of interest by targeting a population of full-length transcripts to enhance coverage by loading individual size fractions on single SMRTcells. More specifically, the SageELF electrophoretic lateral fractionator instrument was used to separate independent fractions of library where isoforms that are 0 – 1kbp, 1kbp – 2kbp, 2kbp - 3kbp and 3kbp – 50kbp were split into independent SMRTbell libraries for sequencing so that larger isoforms weren't detrimentally dominated by smaller isoform library molecules during the sequencing process. Finally, samples were sequenced using 6-hr movie collection on the PacBio RSII sequencer with two SMRTcells per cDNA size fraction.
identifiers.namespace: GEO
identifiers.id: GSM2891092