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Status |
Public on Sep 06, 2022 |
Title |
THP-1 ORF FUT2 rep1 |
Sample type |
SRA |
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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
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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
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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.
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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
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Library strategy |
RNA-Seq |
Library source |
transcriptomic |
Library selection |
cDNA |
Instrument model |
Illumina HiSeq 2000 |
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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
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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) |
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Relations |
BioSample |
SAMN19370065 |
SRA |
SRX11006951 |