|
| Status |
Public on Jul 01, 2018 |
| Title |
5067 F 131 |
| Sample type |
RNA |
| |
|
| Source name |
cecal tissue
|
| Organism |
Mus musculus |
| Characteristics |
gender: F
animal id: 5067 F 131
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Each dissected cecum was filled with a solution of 1.5mM KCL/96 mM NaCl/27mM NaCitrate/8mM KH2PO4/5.6 mM Na2HPO4, pH7.3. were incubated at 37°C after which the intestines were rinsed and filled with Phosphate-buffered saline/1.5 mM EDTA/0.5 mM dithiothreitol. They were incubated at 37°C in a conical centrifuge tube. After an incubation time of 15 minutes, the tube was centrifuged at 900 x g. The resulting pellet was rinsed 3x in Phosphate-buffered saline (centrifuged at 900 x g for 5 minutes after each rinse). The resulting cell pellet was suspended in Trizol (Invitrogen) and RNA was purified by column chromatography as specified by the manufacturer’s protocol. Purified total RNA was fluorescently labeled with riboGreen (Invitrogen) and quantitated using a Spectamax Gemini XPS spectrofluorometer (Molecular Devices). RNA was then prepared for assay by diluting to 50 ng/µl in RNase free water and transferring to a clean 1.5 ml assay tube.
|
| Label |
Cy3
|
| Label protocol |
All assay methods conformed to the exact protocol listed in the Whole-Genome Gene Expression Assay Manual. Diluted RNA (11 µl, ~500ng) was converted to biotinylated cRNA using the Illumina TotalPrep RNA Amplification Kit (Ambion). First, single-stranded cDNA was synthesized from the total RNA using the T7 Oligo(dT) primer, then converted to double-stranded cDNA using the combination of DNA polymerase and RNase H. Second, the cDNA was transcribed to biotinylated cRNA using the T7 enzyme and biotin-labled NTP’s.
|
| |
|
| Hybridization protocol |
The resulting cRNA was column purified, quantitated, and diluted to 150 ng/µl in RNase free water. The sample was then applied to a BeadChip (Illumina MouseWG-6 v2 BeadChip) and hybridized overnight at 58ºC to allow the cRNA to anneal to the oligonucleotides corresponding to their specific gene. The BeadChips were then washed, blocked with E1, and fluorescently labeled with streptavidin-Cy3
|
| Scan protocol |
BeadChips were dried by spinning 300 rpm for 4 min in a benchtop centrifuge and imaged using the Illumina BeadArray Reader
|
| Data processing |
Image data obtained from the BeadArray Reader was analyzed using BeadStudio version 3.0.19 with Gene Expression module 3.0.14. Rank invariant normalization was used with no background subtraction. We filtered the probes that targeted polymorphic sequence due to the bias introduced by microarray probe-target variation in genetic studies and the high density of mouse polymorphisms in the CC. The probes on the Ilumina Mouse WG-6v2.0 Bead Chip were tested by comparing the sequenced genomes from Sanger Wellcome Trust (https://www.sanger.ac.uk/) 78 to determine if they contained a SNP between the strains. 8,972 probes were removed due to SNPs in one of the eight strains and 36,308 probes remained for subsequent QTL mapping and genetic correlation analysis.
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| |
|
| Submission date |
Mar 22, 2017 |
| Last update date |
Jul 01, 2018 |
| Contact name |
Jason A Bubier |
| E-mail(s) |
jbubier@jax.org
|
| Phone |
2072886000
|
| Organization name |
The Jackson Laboratory
|
| Lab |
Chesler Lab
|
| Street address |
600 Main St
|
| City |
Bar Harbor |
| State/province |
ME |
| ZIP/Postal code |
04609 |
| Country |
USA |
| |
|
| Platform ID |
GPL6887 |
| Series (1) |
| GSE96924 |
Cecal mRNA expression in Collaborative Cross Breeding Population |
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