|
| Status |
Public on Sep 20, 2015 |
| Title |
Ring1B I53A/I53A rep1 |
| Sample type |
RNA |
| |
|
| Source name |
E14Tg2a-derived Ring1B I53A/I53A ESCs replicate 1
|
| Organism |
Mus musculus |
| Characteristics |
genotype: Ring1B I53A/I53A
gender: male
strain: 129P2/OlaHsd
cell type: ESC
|
| Growth protocol |
ES cells were grown in GMEM supplemented with 10% fetal bovine serum (FBS), 1,000 units/ml LIF, non-essential amino acids, sodium pyruvate, 2-β-mercaptoethanol and L-glutamine.
|
| Extracted molecule |
polyA RNA |
| Extraction protocol |
RNA was prepared using RNeasy mini kit (Qiagen) according to manufacturer’s protocol, including a DNaseI (Qiagen) treatment for 15min at room temperature
|
| Label |
Cy3
|
| Label protocol |
1ug of purified RNA was mixed with RNA standards (One Colour RNA Spike-In Kit; Agilent – 5188-5282) and the mixture labelled with cyanine 3 (Cy3) using the Amino Allyl MessageAmp™ II with Cy™3 kit (Ambion; AM1795) according to manufacturer’s instructions.
|
| |
|
| Hybridization protocol |
600ng of Cy3-labelled cRNA was fragmented at 60°C for 30 minutes in a reaction volume of 250 ul containing 1x Agilent fragmentation buffer and 2x Agilent blocking agent following the manufacturers instructions. On completion of the fragmentation reaction, 250 il of 2x Agilent hybridization buffer was added to the fragmentation mixture and hybridized to a SurePrint G3 Mouse GE 8x60K microarray (Agilent; G4852A) for 17 hours at 65°C in a rotating Agilent hybridization oven. After hybridization, microarrays were washed 2x1 minute at room temperature with GE Wash Buffer 1 (Agilent) and 2x1 minute with 37°C GE Wash buffer 2 (Agilent), then dried immediately by brief centrifugation.
|
| Scan protocol |
The microarrays were scanned on a Nimblegen scanner at 2um resolution and 25% gain to generate single channel TIFF images.
|
| Description |
Gene expression of E14Tg2a-derived Ring1B I53A/I53A mESCs
|
| Data processing |
The scanned images were analyzed with Feature Extraction Software (Agilent) using default parameters (protocol GE1_1105_Oct12 and Grid: 028005_D_F_20131202) to obtain non-background subtracted and spatially detrended processed signal intensities. Duplicate probe intensity values were replaced with their arithmetic mean. Normalised values were produced using the limma package (R/Bioconductor) by quantile normalisation across all samples. The output file provides log2 normalised intensity values for the 55,681 unique probes (excluding control probes), available in sample table.
Differential Expression Analysis: The limma package was used to calculate fold-changes and p-values for differential expression using the empirical Bayes statistics for differential expression (eBayes) framework. To assign probes to gene names we created a custom probe annotation by downloading a list of mouse transcripts from Ensembl, and identifying probes which map to any transcript with no more than one mismatch, deletion, or insertion. We then used the probe sequences as input to the online version of RepeatMasker using the cross_match search engine and setting “Speed/Sensitivity” to “slow” and “DNA source” to “mouse”. Probes were only included in subsequent analysis if they mapped to transcripts from exactly one gene, and had less than 50 % repetitive sequence as identified by RepeatMasker. The Benjamini-Hochberg method was used to adjust p-values for multiple testing. A probe was considered differentially expressed if it had a fold change greater than 2 or less than 0.5 and an adjusted p-value of less than 0.05. Three biological replicates were used per sample. Final processed data is included as a supplementary file (‘processed_data.txt’, available on the series record) which contains the following information: Sample independent information ProbeName (Agilent probe ID) sequence (Agilent probe sequence) transcript (Ensembl transcript ID/s) ensGene (Ensembl gene name) ens.chr (chromosome/s) ens.TSS.mm10 (transcription start site/s - mm10) SystematicName (Gene symbol or gene location – mm10). Sample dependent information (X denotes e14, KO_G6 or I53A) X.logFC (log2 (intensityX/intensityWT)) X.pval (p value). X.padj (p value following BH multiple testing correction). X (log2 normalised intensity value for X) X.mean (mean log2 normalised intensity value for X) X.up (genes which are upregulated in X vs. e14s) X.down (genes which are downregulated in X vs. e14s)
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| |
|
| Submission date |
Jun 12, 2015 |
| Last update date |
Sep 20, 2015 |
| Contact name |
Rob S Illingworth |
| E-mail(s) |
robert.illingworth@ed.ac.uk
|
| Phone |
01316519640
|
| Organization name |
The University of Edinburgh
|
| Department |
Centre for regenerative Medicine
|
| Lab |
Illingworth
|
| Street address |
Centre for Regenerative Medicine
|
| City |
Edinburgh |
| State/province |
Midlothian |
| ZIP/Postal code |
EH16 4UU |
| Country |
United Kingdom |
| |
|
| Platform ID |
GPL10787 |
| Series (2) |
| GSE69824 |
Catalytically inactive Ring1B maintains near wildtype levels of gene expression in mESCs |
| GSE69978 |
Loss of Ring1B catalytic activity causes a pronounced reduction in H3K27me3 deposition yet minimally disrupts the expression of target genes |
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