|
| Status |
Public on Oct 25, 2016 |
| Title |
set_2 K562 500 nM LP99 Treatment Replicate 3 |
| Sample type |
RNA |
| |
|
| Source name |
K562 Cells
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: K562
treatment: LP99
|
| Treatment protocol |
Cells were seeded the day prior to treatment at 2x105 per ml. Treatments were performed so that a final concentration of 0.1 % DMSO (Cat.#D1435; Sigma) was achieved and cells were incubated with DMSO or 500 nM (final concentration) of test compound (BSP, JQ1, LP99, GSK2801, iCBP112 or OF1) for 6 h prior to isolation of RNA.
|
| Growth protocol |
Human cell lines (K562 and MV-4-11) were obtained from ATCC and the Leibnitz Institute DSMZ-German Collection of Microorganisms and cell cultures (www.dsmz.de). Cell lines were cultured in RPMI-1640 medium (Cat.#61870-044, Gibco) containing 10% fetal calf serum (Cat.#2-01F10-I, BioConcept), 100 U/ml penicillin and 100 U/ml streptomycin (Cat.#15140-122, Gibco). Cells were grown at 37 °C in a humidified cabinet at 5 % CO2 (Heraeus Function Line).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated using a standard TRIzol (Invitrogen) protocol and prepared using RNeasy columns (Cat.#74106 plus; Qiagen). RNA was quantified using a Nanodrop spectrophotometer (model ND1000; Thermo Fisher) and integrity assessed on a BioAnalyzer (2100; Agilent Laboratories, USA). All samples had a RNA Integrity Number (RIN) ≥ 9.
|
| Label |
biotin
|
| Label protocol |
mRNA samples were processed using the Illumina TotalPrep-96 RNA Amplification Kit followed by the Illumina Whole-Genome Gene Expression Direct Hybridisation Assay
|
| |
|
| Hybridization protocol |
Standard Illumina hybridization protocol on Illumina HumanHT-12 v4 beadchips
|
| Scan protocol |
Standard Illumina scanning protocol on an Illumina iScan Scanner. The Illumina GenomeStudio (v.1.9.0, Illumina Inc.) was used to generate bead files
|
| Description |
SAMPLE 12
K562 500 nM LP99 Treatment Replicate 3
|
| Data processing |
GenomeStudio data were processed in R (v.3.2) (Ihaka and Gentleman, 1996) using Bioconductor (v.3.1) (Gentleman et al., 2004) and the lumi package (v.2.20.2) (Du et al., 2008). Quality controls were carried out using the arrayQualityMetrics package (v.3.24.0) (Kauffmann et al., 2009) taking into account array intensity distributions, distance between arrays and variance mean-dependence. Principal component analysis was used to decide which arrays to process together. Background correction followed by variance stabilizing transform (Lin et al., 2008) and quantile between microarrays normalization were carried out with the lumi package. From the 47231 probe sets available on the HumanHT12 V4 chip, removal of un-expressed probes resulted in 24019 probesets. A linear model was applied employing the limma package (v.3.24.13) (Ritchie et al., 2015) followed by empirical Bayesian analysis to determine differential expression between not-treated and treated samples. Genes were considered differentially expressed if the adjusted P-value, calculated using the Benjamini–Hochberg method (Benjamini and Hochberg, 1995) in order to minimize false discovery rate, was less than 0.05 and the mean level of expression was greater than 1.5-fold.
|
| |
|
| Submission date |
Mar 02, 2016 |
| Last update date |
Oct 25, 2016 |
| Contact name |
Panagis Filippakopoulos |
| E-mail(s) |
panagis.filippakopoulos@gmail.com
|
| Organization name |
Oxford University
|
| Department |
Nuffield Department of Medicine
|
| Lab |
Structural Genomics Consortium
|
| Street address |
ORCRB - Roosevelt Drive
|
| City |
Oxford |
| State/province |
Oxfordshire |
| ZIP/Postal code |
OX3 7DQ |
| Country |
United Kingdom |
| |
|
| Platform ID |
GPL10558 |
| Series (2) |
| GSE78829 |
Promiscuous targeting of bromodomains by Bromosporine identifies BET proteins as master regulators of primary transcription response in leukemia [set2] |
| GSE78830 |
Promiscuous targeting of bromodomains by Bromosporine identifies BET proteins as master regulators of primary transcription response in leukemia |
|
