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Sample GSM5628277 Query DataSets for GSM5628277
Status Public on Oct 16, 2021
Title HT29_ORF_Empty_5
Sample type RNA
 
Source name HT-29 Empty transduction, UniqueID 5
Organism Homo sapiens
Characteristics cell_line: HT-29
orf: Empty
uniqueid: 5
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 12.6379150385286
A_37_P136110 4.44713238430854
A_24_P904903 9.69391215205947
A_37_P136602 6.82860037421865
A_37_P458316 11.5371163873204
A_37_P136604 9.35147666573888
A_37_P136607 11.0426439376736
A_37_P360319 10.8977608545989
A_37_P136600 2.13770097890542
A_37_P136605 10.9835412528779
A_37_P136601 3.7489442913576
A_37_P136611 11.8365013022089
A_37_P136603 7.96080046963869
A_37_P136610 11.31825190908
A_37_P136599 4.32322694792934
A_37_P136608 10.639709228399
A_23_P71624 2.1670751343696
A_37_P096846 3.81031745667943
A_37_P096849 5.65519215664888
A_37_P096841 3.54221550211721

Total number of rows: 27439

Table truncated, full table size 756 Kbytes.




Supplementary file Size Download File type/resource
GSM5628277_SG12024163_255950510080_S001_GE1_107_Sep09_1_1.txt.gz 3.1 Mb (ftp)(http) TXT
Raw data provided as supplementary file
Processed data included within Sample table

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