|
| Status |
Public on Sep 22, 2009 |
| Title |
R.lck2.vs.wt1 dye swap |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
lck-deficient regulatory T cells
|
| Organism |
Mus musculus |
| Characteristics |
Strain: C57BL/6
Gender: male
Age: 12 weeks
Tissue: pooled spleen and lymph nodes
|
| Treatment protocol |
sorted lymphocytes
|
| Growth protocol |
primary cells
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was purified with the RNEasy mini kit (Qiagen) with on-column DNAse treatment according to manufacturer's instructions. Total RNA quality was assessed using a Pico Chip on an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto)
|
| Label |
Cy3
|
| Label protocol |
AminoAllyl-modified amplified RNA (aRNA) was prepared from approximately 1μg of total RNA using the AminoAllyl Message Amp II kit (Ambion). 15μg of this aRNA in 0.05 M sodium bicarbonate pH9.0 were then coupled to N-hydroxysuccinimidyl esters of Cy3 or Cy5 dyes (CyScribe, Amersham Biosciences) for 90 minutes in the dark. The labelled aRNA was neutralized with 100mM sodium acetate pH5.5 and purified using the RNA clean-up kit-25 (Zymo Research). Immediately prior to hybridization, labelled aRNA samples were fragmented (Ambion Fragmentation buffer, Ambion) and denatured at 95ºC for 10 minutes.
|
| |
|
| Channel 2 |
| Source name |
wt regulatory T cells
|
| Organism |
Mus musculus |
| Characteristics |
Strain: C57BL/6
Gender: male
Age: 12 weeks
Tissue: pooled spleen and lymph nodes
|
| Treatment protocol |
sorted lymphocytes
|
| Growth protocol |
primary cells
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was purified with the RNEasy mini kit (Qiagen) with on-column DNAse treatment according to manufacturer's instructions. Total RNA quality was assessed using a Pico Chip on an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto)
|
| Label |
Cy5
|
| Label protocol |
AminoAllyl-modified amplified RNA (aRNA) was prepared from approximately 1μg of total RNA using the AminoAllyl Message Amp II kit (Ambion). 15μg of this aRNA in 0.05 M sodium bicarbonate pH9.0 were then coupled to N-hydroxysuccinimidyl esters of Cy3 or Cy5 dyes (CyScribe, Amersham Biosciences) for 90 minutes in the dark. The labelled aRNA was neutralized with 100mM sodium acetate pH5.5 and purified using the RNA clean-up kit-25 (Zymo Research). Immediately prior to hybridization, labelled aRNA samples were fragmented (Ambion Fragmentation buffer, Ambion) and denatured at 95ºC for 10 minutes.
|
| |
|
| |
| Hybridization protocol |
Hybridizations to Mouse Exonic Evidence Based Oligonucleotide (MEEBO) spotted microarrays were performed at 55˚C for 48 hours in Slide Hybridization buffer (Ambion). A total of 16 arrays were used to analyze regulatory and memory subsets from eight mice (two arrays per littermate pair, including dye swaps, were used for each comparison).
|
| Scan protocol |
After hybridization, the arrays were washed and scanned using an Axon 4000B laser scanner (Molecular Devices). Image analysis was performed using Spotreader (Niles Scientific).
|
| Description |
Amplified total RNA was hybridized to MEEBO arrays
|
| Data processing |
The ‘print-tip loess’ normalization was used to correct for within-array dye and spatial effects and single channel quantile normalization was used to facilitate comparison between arrays. Functions in the library marray Norm of the R/Bioconductor package were used to perform these normalizations.
|
| |
|
| Submission date |
Nov 18, 2008 |
| Last update date |
Sep 22, 2009 |
| Contact name |
Mark Klinger |
| E-mail(s) |
mark.klinger@ucsf.edu
|
| Phone |
415-502-5432
|
| Organization name |
University of California, San Francisco
|
| Department |
Microbiology and Immunology
|
| Lab |
Nigel Killeen
|
| Street address |
513 Parnassus Avenue, Box0414
|
| City |
San Francisco |
| State/province |
CA |
| ZIP/Postal code |
94122 |
| Country |
USA |
| |
|
| Platform ID |
GPL7656 |
| Series (1) |
| GSE13645 |
Transcriptional profiling of p56lck-deficient regulatory and memory T cells |
|
