|
| Status |
Public on Jan 18, 2012 |
| Title |
homozygous congenic 2 |
| Sample type |
RNA |
| |
|
| Source name |
left and right striatum
|
| Organism |
Mus musculus |
| Characteristics |
batch: batch 2
genotype: homozyygous for A/J in congenic region
Sex: female
age (days old): 57
|
| Treatment protocol |
Mice were housed as same-sex littermates, 2-5 per cage. Mice were naïve at the time of rapid decapitation for brain tissue harvesting.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was extracted using Trizol® (Invitrogen, Carlsbad, CA) and chloroform and then precipitated with ethanol and resuspended. The solution was then passed through RNeasy® columns (Qiagen), quality checked using the Agilent 2100 Bioanalyzer (Santa Clara, CA),
|
| Label |
biotin
|
| Label protocol |
Precision Biomarker Resources processed the RNA and scanned the arrays. Here is the protocol manual with all of the details for the amplification: http://tools.invitrogen.com/content/sfs/manuals/cms_064619.pdf. Briefly, we started with 100ng of total RNA, subjected it to first strand and second strand cDNA generation followed by 16hr IVT amplification making cRNA. The cRNA was bead purified and then 10µg was put into the second cycle sscDNA reaction. 5.5µg of sscDNA was then fragmented and end labeled with biotin.
|
| |
|
| Hybridization protocol |
The details for the hyb/wash/stain are in the Affy protocol: http://media.affymetrix.com/support/downloads/manuals/wt_term_label_ambion_user_manual.pdf. Briefly, the hybridization cocktail was made with the Affy Hyb buffer, DMSO, Oligo B2 and 20X eukaryotic controls and hybridized to the gene arrays for 17hrs at 45C. The arrays were then washed and stained with SAPE (Streptavidin Phycoerythrin binds to the biotin) and scanned.
|
| Scan protocol |
Affymetrix GeneChip Scanner 3000 7G. Affymetrix GeneChip Command Console (AGCC) software generated the raw data files including the CEL files.
|
| Description |
11-1831_CB_(MoGene-1_0-st-v1).CEL
not littermates of the other genotype
|
| Data processing |
Data were processed using both Affymetrix Gene Expression Console Software and Affymetrix Powertools robust multichip average (RMA) analysis. Both gave the same results. However, prior to generating RMA values, we eliminated 145 probes from analysis using the Affymetrix powertools kill list option. We provide the text file named kill list that we used for probe masking. After generating RMA values, we then corrected for batch effects (2 batches) using the ComBat.R script which is also provided. Thus, the RMA values provided on the matrix template are those obtained following probe masking and following correction for batch effects.
|
| |
|
| Submission date |
Jan 17, 2012 |
| Last update date |
Jan 18, 2012 |
| Contact name |
Camron D Bryant |
| E-mail(s) |
[email protected]
|
| Phone |
(773) 834-0604
|
| Fax |
(773) 834-0505
|
| Organization name |
The University of Chicago
|
| Department |
Human Genetics
|
| Lab |
Abraham Palmer
|
| Street address |
920 E. 58th Street, CLSC 501
|
| City |
Chicago |
| State/province |
IL |
| ZIP/Postal code |
60616 |
| Country |
USA |
| |
|
| Platform ID |
GPL6246 |
| Series (1) |
| GSE35139 |
Congenic dissection of a major QTL for methamphetamine sensitivity implicates epistasis |
|
