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Sample GSM4590985 Query DataSets for GSM4590985
Status Public on Sep 15, 2021
Title HeLa cell culture - CDT HA - Rep3 [HA_3]
Sample type RNA
 
Source name HeLa cell culture - CDT HA
Organism Homo sapiens
Characteristics cell line: HeLa
condition: CDT HA
exposure time: 8 weeks
rna integrity number: 10
Treatment protocol One day after seeding, cells were exposed to 0.25 ng/ml of WT or HA CDT for 48 H. For chronic exposure, individual clones were selected, the rest of the cells were pooled. CDT was added at each passage (every 2-3 days) for 8 weeks.
Growth protocol HeLa cells were were cultured in Dulbecco’s Modified Eagle Medium (DMEM, Life Technologies), supplemented with 10% heat-inactivated fetal bovine serum (FBS, Gibco) and 1% antibiotics (penicillin/streptomycin). Cells were plated at a density of 150000 cells per well in 6 wells plate
Extracted molecule total RNA
Extraction protocol Total RNA was extracted with TRIzol reagent (Invitrogen, Carlsbad, CA, USA) mRNA were extracted and the quality of these samples was assessed (Agilent RNA 6000 Nano Kit Quick, Agilent Bioanalyzer 2100); RNA Integrity Number (RIN) of these mRNA was superior to 9.8.
Label Cy3
Label protocol For each sample, Cyanine-3 (Cy3) labeled cRNA was prepared from 200 ng of total RNA using the One-Color Quick Amp Labeling kit (Agilent Technologies, Santa Clara, CA) according to the manufacturer's instructions, followed by Agencourt RNAClean XP (Agencourt Bioscience Corporation, Beverly, Massachusetts) purification. Dye incorporation and cRNA yield were checked using Dropsense™ 96 UV/VIS droplet reader (Trinean, Belgium).
 
Hybridization protocol 600 ng of Cy3-labelled cRNA (specific activity >6 pmol Cy3/µg cRNA) was fragmented at 60°C for 30 minutes in a reaction volume of 25 µl containing 10x Agilent fragmentation buffer and 25x Agilent blocking agent following the manufacturer's instructions (Agilent Technologies, Santa Clara, CA). On completion of the fragmentation reaction, 25 µl of 2x Agilent hybridization buffer was added to the fragmentation mixture and hybridized to SurePrint G3 Human GE v2 microarray (8X60K, Design 039494) enclosed in Agilent SureHyb-enabled hybridization chambers for 17 hours at 65°C in a rotating Agilent hybridization oven. After hybridization, microarrays were washed sequentially in Wash buffer 1 (Agilent Technologies, 1 min) and Wash buffer 2 (Agilent Technologies, 37°C, 1 min).
Scan protocol Slides were scanned immediately after washing on a Agilent G2505C Microarray Scanner with Agilent Scan Control A.8.5.1 software
Data processing The scanned images were analyzed with Feature Extraction Software 10.10.1.1 (Agilent Technologies, Santa Clara, CA) using default parameters (protocol GE1_1010_Sep10 and Grid: 039494_D_F_20120628). All subsequent data analyses were done under R (www.r-project.org) using packages of Bioconductor (www.bioconductor.org). Raw data (median of pixels intensity) were imported into R using the read.maimages function from the limma package with the following weight function (assigning a weight of 1 or 0 to each spot): myfunw<-function(x) {okType<-x$ControlType==0; okFoundGreen<-x$gIsFound==1; okPos=x$gIsPosAndSignif==1; okWellAbove<- x$gIsWellAboveBG==1; as.numeric(okType & okFoundGreen & okPos & okWellAbove);} We selected the spots with a minimal weight of 1 for 12 out of 15 microarrays or with a minimal weight of 3 per group from at least one experimental group. At this step, 32772 spots out of 62976 were selected. Data were then stored in an ExpressionSet object and normalized by the quantile method using the normalize.quantiles function from the preprocessCore R library. Replicated probes on the array (identical ProbeName) were resolved by taking the median normalized signal of each set of replicated probes. The resulting matrix has 26516 rows each corresponding to a unique ProbeName (provided as data Matrix).
 
Submission date Jun 04, 2020
Last update date Sep 15, 2021
Contact name Yannick Lippi
E-mail(s) yannick.lippi@inrae.fr
Organization name INRAE
Department GeT-TRiX facility
Lab TOXALIM UMR1331
Street address 180 chemin de tournefeuille
City Toulouse
ZIP/Postal code 31300
Country France
 
Platform ID GPL17077
Series (1)
GSE151792 Transcriptional adaptation to chronic exposure to Cytolethal Distending Toxin in HeLa cells

Data table header descriptions
ID_REF
VALUE log2 normalized signal

Data table
ID_REF VALUE
A_23_P117082 12.67355215
A_33_P3246448 5.369256209
A_21_P0000509 15.92402746
A_21_P0000744 10.0414579
A_24_P215804 5.775640156
A_23_P110167 12.26972628
A_33_P3211513 7.458813963
A_33_P3414202 7.188765094
A_33_P3316686 7.802188899
A_33_P3300975 8.512000733
A_33_P3263061 12.4613741
A_24_P278460 9.601037388
A_21_P0014651 6.387937603
A_24_P286898 6.307184429
A_23_P340890 10.47724133
A_21_P0010885 11.82048551
A_23_P89762 7.0987496
A_23_P109034 9.481391957
A_21_P0013603 5.585010685
A_33_P3261031 5.997768396

Total number of rows: 26516

Table truncated, full table size 653 Kbytes.




Supplementary file Size Download File type/resource
GSM4590985_US10463851_253949436680_S01_GE1_1010_Sep10_1_2.txt.gz 12.3 Mb (ftp)(http) TXT
Processed data included within Sample table

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