|
| Status |
Public on Oct 23, 2013 |
| Title |
Caecum ss cDNA |
| Sample type |
mixed |
| |
|
| Channel 1 |
| Source name |
Caecum
|
| Organism |
Mus musculus |
| Characteristics |
genotype: wild-type
sample type: ss cDNA
strain: Bl6/C57
tissue: Caecum
age: E13.5
|
| Treatment protocol |
RNA was depleted of rRNA 18S and 28S using RiboMinus Human/Mouse Transcriptome Isolation kit (Affymetrix). Using T7-(N)6 primers, RiboMinus-enriched RNA was stepwise reverse transcribed to cRNA (SuperScript II) and to double-stranded (ds) cDNA (DNA Polymerase I). cDNA was further amplifed by in vitro transcription to cRNA and reverse transcribed again to single-stranded (ss) cDNA (Superscript II) with incorporation of deoxyuridine using the GeneChip Whole Transcript Amplified Double-Stranded Target Assay kit (Affymetrix). For ss cDNA tiling array, cDNA was treated with NaOH 1M and blocked with HCl 1M. For ds cDNA tiling array, cDNA was reverse transcribed to ds cDNA (DNA Polymerase I) with incorporation of deoxyuridine. Both ss and ds cDNAs were fragmented with apurinic / apyrimidinic endonucleases and uracil DNA glycosylases and labeled with terminal deoxynucleotidyl transferase using GeneChip WT Double- Stranded DNA Terminal Labeling Kit (Affymetrix). The profile of all different steps was analysed with a Bioanalyser. cDNAs were hybridized to custom-made tiling arrays (Soshnikova and Duboule, 2009). Tiling arrays were processed according to manufacturer instructions. Transcript profiling experiment was repeated once for each sample, except for del(1-4i) and del(9-11) caeca.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Caeca were dissected from E13.5 embryos and and stored in RNAlater reagent (QIAGEN) until genotyped. After disruption with PT1200E Polytron (Kinematica), RNA was isolated using the RNeasy minikit (QIAGEN).
|
| Label |
biotin
|
| Label protocol |
standard Affymetrix protocol
|
| |
|
| Channel 2 |
| Source name |
gDNA
|
| Organism |
Mus musculus |
| Characteristics |
age: E13.5
|
| Treatment protocol |
RNA was depleted of rRNA 18S and 28S using RiboMinus Human/Mouse Transcriptome Isolation kit (Affymetrix). Using T7-(N)6 primers, RiboMinus-enriched RNA was stepwise reverse transcribed to cRNA (SuperScript II) and to double-stranded (ds) cDNA (DNA Polymerase I). cDNA was further amplifed by in vitro transcription to cRNA and reverse transcribed again to single-stranded (ss) cDNA (Superscript II) with incorporation of deoxyuridine using the GeneChip Whole Transcript Amplified Double-Stranded Target Assay kit (Affymetrix). For ss cDNA tiling array, cDNA was treated with NaOH 1M and blocked with HCl 1M. For ds cDNA tiling array, cDNA was reverse transcribed to ds cDNA (DNA Polymerase I) with incorporation of deoxyuridine. Both ss and ds cDNAs were fragmented with apurinic / apyrimidinic endonucleases and uracil DNA glycosylases and labeled with terminal deoxynucleotidyl transferase using GeneChip WT Double- Stranded DNA Terminal Labeling Kit (Affymetrix). The profile of all different steps was analysed with a Bioanalyser. cDNAs were hybridized to custom-made tiling arrays (Soshnikova and Duboule, 2009). Tiling arrays were processed according to manufacturer instructions. Transcript profiling experiment was repeated once for each sample, except for del(1-4i) and del(9-11) caeca.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Caeca were dissected from E13.5 embryos and and stored in RNAlater reagent (QIAGEN) until genotyped. After disruption with PT1200E Polytron (Kinematica), RNA was isolated using the RNeasy minikit (QIAGEN).
|
| Label |
biotin
|
| Label protocol |
standard Affymetrix protocol
|
| |
|
| |
| Hybridization protocol |
standard Affymetrix protocol
|
| Scan protocol |
standard Affymetrix protocol
|
| Description |
ds cDNA normalized with ss cDNA
|
| Data processing |
cDNA array data were normalized with ds cDNA array replicates for ss cDNA, to genomic DNA array replicates for ds cDNA, and scaled to feature intensity of 10 using TAS software (Affymetrix). PM-MM pairs mapping within a sliding window of 75 bp was used.
bar files of normalized signals
|
| |
|
| Submission date |
Feb 15, 2013 |
| Last update date |
Oct 23, 2013 |
| Contact name |
Marion LELEU |
| Organization name |
EPFL
|
| Department |
School of Life Sciences
|
| Lab |
Laboratory of developmental genomics
|
| Street address |
EPFL-SV-ISREC-UPDUB- Station 19
|
| City |
Lausanne |
| ZIP/Postal code |
1015 |
| Country |
Switzerland |
| |
|
| Platform ID |
GPL14183 |
| Series (2) |
| GSE44362 |
Transcription landscape at the HoxD locus in developing caeca |
| GSE45242 |
HoxD locus |
|
