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Sample GSM5343872 Query DataSets for GSM5343872
Status Public on Sep 06, 2022
Title THP-1 ORF FUT2 rep1
Sample type SRA
 
Source name THP-1 cells
Organism Homo sapiens
Characteristics cell type: Monocytic leukemia
overexpressed gene: FUT2
sampleID: FUT2_1214
plasmid: pLVX-EF1a-IRES-PURO/eGFP
infection date: 13-01-2016
days in culture: 18
sequencing batch: B
Treatment protocol THP-1 cells were transduced in triplicate via spinoculation by open reading frames (ORFs) for 42 genes cloned into a modified GATEWAY® compatible polycistronic lentiviral expression vector, pLVX-EF1a-IRES-PURO/eGFP
Growth protocol THP-1 were maintained at 37°C with 5% atmospheric CO2 in RPMI 1600 supplemented with 10% FBS fetal bovine serum, 1% penicillin/streptomycin (Thermo Fisher Scientific) and 0.05 mM beta-mercaptoethanol  (Invitrogen). THP-1 were grown for an average of 7 days (with a range of 4-16 days) to select successfully transduced cells and reach 1x106 cells/ml (3-5 x106 cells in total) before RNA extraction.
Extracted molecule total RNA
Extraction protocol Total RNA was extracted from stably transduced THP-1 cultures using the RNeasy Plus Mini kit (Qiagen) according to manufacturer’s protocol. The RNA samples were quantified, and quality controlled using an Agilent RNA 6000 Nano kit (Agilent) on an Agilent Bioanalyzer 2100 system. Samples with RNA Integrity Number (RIN) below 8 were discarded.
RNA samples were transformed into barcoded DNA libraries using TruSeq Stranded mRNA library preparation kits (Illumina), which were then paired-end sequenced, generating 2x100bp reads, using an Illumina HiSeq2000 sequencer
 
Library strategy RNA-Seq
Library source transcriptomic
Library selection cDNA
Instrument model Illumina HiSeq 2000
 
Description THP-1 stable cell line overexpressing FUT2 gene / replicate 1
Data processing Illumina HiSeq2000 using HiSeq Control Software
Primary QC analysis was performed using FastQC (v0.11.5; https://www.bioinformatics.babraham.ac.uk/projects/fastqc)
Sequence filtering parameters set by Trimmomatic (v.0.36 ) Primer sequences were filtered out with provided TruSeq sequence data and a quality sliding window of Q over 20 for a 40-nucleotide window; only sequences longer than 70 nucleotides were kept in the filtered FASTQ files. Another FastQC run was performed on all filtered FASTQ files to validate Trimmomatic results.
Filtered FASTQ files were aligned to the human genome with STAR (v. 2.5.3a) using indexes built with STAR from H. sapiens GRCh Build 38 files (reference genome and GTF annotation files) provided by Illumina's iGenome repository.
Default values were selected except for the –quantMode flag that was set to TranscriptomeSAM for downstream quantification. Counts per gene for each sequenced sample were obtained using RSEM (v. 1.2.6)
Gene counts were transformed to FPKM as an approximation of gene relative abundance. Genes were filtered to keep only those with FPKM > 0.3 and count>8 in at least two samples.
Small RNAs and LOC genes without annotations were removed. Quality control of samples was performed, excluding strong outlier samples based on principal component analyses
The log-transformed FPKM were then normalized between samples with cyclicloess (normalizeCyclicloess from sva Bioconductor R package).
RNA-seq and cell culture batches effects were removed using ComBat (sva Bioconductor library v3.18.0).
Genome_build: GRCh 38
Supplementary_files_format_and_content: THP1_overexpression_processed_20210506.txt; tab delimited text file; normalized FPKM table
 
Submission date May 27, 2021
Last update date Sep 06, 2022
Contact name John D Rioux
Organization name Montreal Heart Institute
Department Research Center
Lab Genetics and genomic medecine of Inflammation
Street address 5000 Bélanger St.
City Montréal
State/province QC
ZIP/Postal code H1T 1C8
Country Canada
 
Platform ID GPL11154
Series (1)
GSE175685 Transcriptomic profiling of THP-1 stable cell lines overexpressing genes associated with inflammatory bowel diseases (IBD)
Relations
BioSample SAMN19370065
SRA SRX11006951

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

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