GEO Logo
   NCBI > GEO > Accession DisplayHelp Not logged in | LoginHelp
GEO help: Mouse over screen elements for information.
Sample GSM2891095 Query DataSets for GSM2891095
Status Public on Dec 16, 2017
Title siNT_48-PacBio Iso-Seq
Sample type SRA
Source name CRC cell line SW480
Organism Homo sapiens
Characteristics cell line: SW480
genotype/variation: control (siNT)
experiment: Mono
Treatment protocol 24 hours after seeding, cells were transfected in duplo with small interfering RNA (siRNA) pools against SF3B1 (siGENOME SF3B1 SMARTpool, M-020061-02; Thermo Fisher Scientific, Waltham, USA) and SRSF1 (siGENOME SRSF1 SMARTpool, M-018672-01), according to manufacturer’s recommendations. A final siRNA concentration of 30 nM was obtained using DharmaFECT3 reagent (1:1000 dilution; T-2003-02, Thermo Fisher Scientific). A non-targeting siRNA pool (siGENOME Non-Targeting pool #2, D-001206-14) was used as negative control.
Growth protocol SW480 cells cultured in Dulbecco's modified Eagle's medium (DMEM; Invitrogen, Bleiswijk, The Netherlands) containing 10% fetal bovine serum (FBS; Perbio Science, Etten-Leur, The Netherlands) were maintained in a humidified 5% CO2 atmosphere at 37 °C.
Extracted molecule total RNA
Extraction 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.
Library strategy RNA-Seq
Library source transcriptomic
Library selection cDNA
Instrument model PacBio RS II
Description siRNA mediated down-modulation of NonTargeting control for siSF3B1, RNA and proteins were harvested in 48hours, single experiment performed
Data processing Illumina RNA-seq: Low quality reads and adapter sequences were trimmed by Trimmomatic version 3 to average quality score for a 4-base wide sliding window of 20, both at the beginning and at the end of the sequences (ILLUMINACLIP:TruSeq3-PE.fa:2:30:10, LEADING:20, TRAILING:20, AVGQUAL:20, SLIDINGWINDOW:4:20). Due to the requirements of the further analysis reads were processed to match length of 120bp, shorter reads were discarded and longer reads were trimmed (CROP:120, MINLEN:120). Mapping was performed with the use of STAR aligner version 2.4.2a to the human genome (USCS RefSeq hg19 annotation, as STAR option genomeDir) with the following parameters; outSAMtype: BAM SortedByCoordinate, readFilesCommand: zcat, runThreadN: 28, outSAMattributes: All. Read counts per transcript were obtained with featureCounts from the Subread package v1.5.0-p2 with the UCSC RefSeq hg19 GTF file as reference. Differential splice variants were identified with rMATS version 3.2.5 using UCSC RefSeq hg19 GTF file as annotation in the unpaired analysis type (parameters; len: 120, t: paired, analysis: U). Significant events were extracted (FDR≤0.05) and transformed to a bedGraph. For the Mono experiment additional rMATS analysis was performed with PacBio GFF file as reference (all_samples.chained.gff).
PacBio Iso-seq: Redundant transcripts were removed by first aligning them to the human genome (hg19) with GMAP and collapsing highly similar transcripts predicted across FASTA files from various size fractions with the software cupcake ToFU (v1.3). In these steps both BAM and GTF files were produced for each sample. Samples were chained, to standardize transcript IDs and merge the transcripts from both experiments (all_samples.chained.gff).
Genome_build: hg19
Supplementary_files_format_and_content: TXT files include expression matrix with raw RNA-seq read counts per sample, bed Graphs represent differential splice variants between conditions identified from Illuminna RNA-seq data, GFF files include all transcripts for samples measured by PacBio Iso-Seq
Submission date Dec 15, 2017
Last update date Dec 18, 2017
Contact name Remond JA Fijneman
Phone +31 50 512 1725
Organization name Netherlands Cancer Institute
Department Pathology
Lab Translational Gastrointestinal Oncology
Street address Plesmanlaan 121
City Amsterdam
ZIP/Postal code 1066 CX
Country Netherlands
Platform ID GPL21311
Series (1)
GSE108140 Identification of Differentially Expressed Splice Variants by the Proteogenomic Pipeline Splicify
BioSample SAMN08182060
SRA SRX3474291

Supplementary data files not provided
SRA Run SelectorHelp
Raw data are available in SRA
Processed data are available on Series record

| NLM | NIH | GEO Help | Disclaimer | Accessibility |
NCBI Home NCBI Search NCBI SiteMap