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Sample GSM1533168 Query DataSets for GSM1533168
Status Public on Apr 02, 2015
Title shH1X_Dox_rep1
Sample type RNA
Source name breast cancer cell T47D-MTVL (breast cancer cell line carrying one stably integrated copy of luciferase reporter gene driven by the MMTV promoter)
Organism Homo sapiens
Characteristics cell line: T47D-MTVL
gender: female
tissue: breast cancer ductal carcinoma
dox treatment: Treated
depletion/expression: Histone H1X depletion
expression: Histone H1X shRNA vector
Treatment protocol Initially, a cell line expressing the Dox-responsive KRAB repressor and RedFP (ptTR-KRAB-Red) was generated. Then, this cell line was infected with viruses for expression of the different H1 variants shRNAs (pLVTHM). The inducible knocked-down cell lines were sorted in a FACSvantageSE (Becton Dickinson) for RedFP-positive and GFP-positive fluorescence after 3 days of Dox treatment. Then cells were amplified in the absence of Dox until an experiment was performed. Doxicycline (Sigma) was added at 2.5 µg/ml when indicated. Along a 6-day treatment with Dox, cells were passaged at day 3. Serum-containing media was replaced with serum-free media at day 4 for growth arrest.
Growth protocol Cell lines were grown in RPMI 1640 medium, supplemented with 10% FBS, 2mM L-glutamine, 100 U/ml penicillin, and 100µg/ml streptomycin.
Extracted molecule total RNA
Extraction protocol Total RNA was extracted using High Pure RNA isolation kit (Roche) according to the manufacturer's instructions. cDNA was obtained from 100 ng of total RNA using SuperScript VILO cDNA synthesis (Invitrogen). High RNA integrity was assessed by Bioanalyzer nano 6000 assay with RIN numbers 9.5
Label Cy3
Label protocol For each sample 100 ng of total RNA mixed with 2 ul of 1:10000 Agilent One Color Spike Mix were reversed transcribed into cDNA with a T7 promoter and the cDNA was in vitro transcribed into cRNA in the presence of Cy3-CTP using the Low input quick Amp kit (Agilent). Labelled samples were purified using RNeasy mini spin columns (Qagen) and yield and specific activity wwere assessed by spectrophotometry (Nanodrop) and confirmed to be within specifications (>825 ng and >6 pmol Cy3/ug cRNA). 600 ng of cRNA were preblocked and fragmented in Agilent fragmentation buffer and mixed with Agilent GEx Hybridization mix.
Hybridization protocol Hybridization mix was laid onto each sector of subarray gasket slide and sandwiched against a 8 x 65K format oligonucleotide microarray (Human v1 Sureprint G3 Human GE 8x60k Microarray, Agilent design ID 028004) inside a hybridization chamber which wqs hybridized overnight at 65ºC in a rotisserie oven rotating at 10 rpm for a total of 17 hours. Subsequently array chambers were disassembled submerged in Agilent Gene Expression Buffer 1 and washed 1 minute in another dish with the same solution with a magnetic stirrer at 200 rpm at room temperature, followed by 1 minute in gilent Gene Expression Buffer 2 with a magnetic stirrer at 200 rpm at 37ºC and immediate withdrawal from the solution and air drying.
Scan protocol Fluorescent signal was captured into TIF images with an Agilent scanner using recommended settings (20 bit, 5 um resolution, 100% PMT and 100% laser power) with Scan Control software (Agilent).
Description H1X knock down, T47D-MTVL breast cancer cell line transfected with doxicyclin inducible anti histone H1 shRNA expression vector treated with doxicyclin, replicate 1 of 2
Data processing Signal intensities were extracted into a tabulated text file using Feature Extraction software (Agilent) using the appropriate array configuration and annotation files. The normalized log2intensities were obtained using quantile method with normexp background correction the Bioconductor Limma package in R.
Submission date Oct 28, 2014
Last update date Apr 02, 2015
Contact name Lauro Sumoy
Organization name IGTP
Department High Content Genomics and Bioinformatics
Street address Ctra. Can Ruti, Camí de les escoles s/n
City Badalona
State/province Barcelona
ZIP/Postal code 08916
Country Spain
Platform ID GPL13607
Series (1)
GSE62766 Gene expression of T47D-MTVL human breast cancer cells upon H1X knock-down

Data table header descriptions
VALUE Normalized log2 of Cy3 intensities

Data table
1 15.78004757
2 6.249546359
3 6.304034544
4 9.658363828
5 10.15862723
6 7.377641001
7 12.40913887
8 10.3012671
9 6.859041823
10 7.286851177
11 6.518169319
12 12.0606765
13 9.989566281
14 9.116565748
15 13.41446057
16 6.524899394
17 8.473037318
18 6.354022113
19 6.609647948
20 10.01831888

Total number of rows: 62976

Table truncated, full table size 1091 Kbytes.

Supplementary file Size Download File type/resource
GSM1533168_US10403838_252800412787_S01_GE1_107_Sep09_2_3.txt.gz 12.3 Mb (ftp)(http) TXT
Processed data included within Sample table

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