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Sample GSM5628294 Query DataSets for GSM5628294
Status Public on Oct 16, 2021
Title HT29_ORF_RIT1_179
Sample type RNA
 
Source name HT-29 RIT1 transduction, UniqueID 179
Organism Homo sapiens
Characteristics cell_line: HT-29
orf: RIT1
uniqueid: 179
arraybatch: E1
transductiondate: 2015-02-26
Treatment protocol ORFs for IBD gene candidates were cloned into a GATEWAY® compatible polycistronic lentiviral expression vector, pLVX-EF1a-IRES-PURO/eGFP expressing an enhanced green fluorescent protein (eGFP) and puromycin N-acetyl-transferase fusion protein.
Proliferative HT-29 cells at 50% confluency in 12 well plate (Corning) were transduced in triplicate with the different ORFs (multiplicity of infection) MOI of 40-100 with 8µg/ml of polybrene (Sigma, Cat: H-9268).
Twenty-four hours post-transduction, media was changed and 3ug/ml puromycin (Millipore Sigma) was added.
The appearance and confluence of the cultures were recorded daily, and cells were grown for an average of 8 days (with a range of 5-27 days) to select successfully transduced cells and reach confluence before RNA extraction.
Transduction of the whole set of IBD gene candidate ORFs was performed in batches of about 15 ORFs, each done in triplicate, and including an empty vector control.
Growth protocol The colorectal adenocarcinoma cell line HT-29 (ATCC HTB-38) was maintained at confluence between 20% and 80% at all times in McCoy’s 5A (Wisent, 317-010CL) 10% FBS. All experiments described herein were performed in cell at passages below 25.
Extracted molecule total RNA
Extraction protocol The RNeasy Plus Mini kit (#74036, Qiagen Inc Canada) according to manufacturer’s protocol.
The RNA samples were quantified, and quality controlled using an Agilent RNA 6000 Nano kit (Agilent, Cat No./ID 5067-1511) on 2100 Bioanalyzer system.
Number (RIN) below 8 were discarded; RIN values were routinely in the range of 9.2-10
Label Cy3
Label protocol Agilent One-Color Microarray-Based Exon Analysis Low Input Quick Amp WT Labeling kit using Cyanine 3-CTP label.
 
Hybridization protocol Samples were blocked with Agilent blocking reagent and incubated for 17h at 65C in the Agilent Microarray Hybridization oven
Scan protocol Agilent SureScan Microarray scanner
Data processing We used Limma v3.26.9 and sva v3.18.0 packages (Bioconductor v3.1-3.2) to load the fluorescence intensity files and handle the data into R V3.2.0. Arrays failing Agilent quality control according to the Agilent Feature Extraction software were flagged. Manual quality control of each array was also performed.
Quality control was performed within each batch of arrays. Fluorescence signal was processed to remove local background (bakgroundCorrect, method “normexp”) and then normalized using cyclic loess algorithm.
Batch effects were then corrected using ComBat for known batches and sva for unknown systematic artefacts (both from sva library v3.18.0). Given the experiment was performed in three parts (E1-E4 & P7, E5-E11, E14-E15), the batches from each part were combined first and then combined together.
To reduce impact of residual background, noise and batch effects on very low expression values, the fluorescence intensity values were truncated at 8, and translated down by the same amount.
Duplicated probes were combined, and repeated samples removed. The truncating threshold was determined based on the distribution of negative control probes and detected vs non-detected signals. Data provided includes only samples included in the final dataset.
 
Submission date Oct 15, 2021
Last update date Oct 16, 2021
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 GPL30867
Series (1)
GSE186001 Functional screen of Inflammatory bowel disease genes reveals key epithelial functions: Agilent Targeted Dataset

Data table header descriptions
ID_REF
VALUE background corrected,cyclic loess normalized batch effect removed (using ComBat) log2-transformed data

Data table
ID_REF VALUE
A_37_P136616 13.1602706627111
A_37_P136110 4.9325350814054
A_24_P904903 9.8313663979093
A_37_P136602 6.89823939448169
A_37_P458316 10.8420147389889
A_37_P136604 9.74018676059795
A_37_P136607 11.6137146450201
A_37_P360319 11.4728797384372
A_37_P136600 2.59873199604696
A_37_P136605 11.1385957798197
A_37_P136601 4.46394615634397
A_37_P136611 12.391806909566
A_37_P136603 8.28646362306973
A_37_P136610 11.5583949047549
A_37_P136599 4.37987797157553
A_37_P136608 11.2376135702833
A_23_P71624 2.20761308910153
A_37_P096846 3.97355624785051
A_37_P096849 6.13564155310635
A_37_P096841 3.58266656288509

Total number of rows: 27439

Table truncated, full table size 756 Kbytes.




Supplementary file Size Download File type/resource
GSM5628294_SG12024163_255950510082_S001_GE1_107_Sep09_1_3.txt.gz 3.0 Mb (ftp)(http) TXT
Raw data provided as supplementary file
Processed data included within Sample table

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