|
| Status |
Public on Sep 15, 2015 |
| Title |
Control ISC biological rep 2 |
| Sample type |
SRA |
| |
|
| Source name |
Control mouse intestinal stem cells
|
| Organism |
Mus musculus |
| Characteristics |
strain: B6;129 mixed background
cell type: Adult intestinal stem cell; proximal 2/3 of intestine
genotype: Cdx2+/+; Lgr5-EGFP-IRES-CREERT2
|
| Treatment protocol |
Mice were injected with 1-2mg Tamoxifen for 4-5 consecutive days to activate tamoxifen-inducible Cre and recombine conditional alleles.
|
| Growth protocol |
Crypts from the proximal 2/3 of mouse small intestines were isolated by scraping off villi and incubating in 5mM EDTA in PBS for 45 minutes at 4°C. Crypts were disaggregated with 4X TrypLE in DMEM for 45 minutes at 37°C and Lgr5-GFP-HI intestinal stem cells were sorted by flow cytometry.
|
| Extracted molecule |
polyA RNA |
| Extraction protocol |
poly-A RNA was extracted with oligo dT magnetic beads (New England Biolabs) followed by DNase treatment with the RNeasy kit (Qiagen).
The Encore Complete RNA-Seq Library System (Nugen) was used, modified for 1/2 volume reactions. 200-450 bp fragments were submitted for sequencing 50 bp single-end reads on an Illumina Hi-Seq 2000 instrument.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2000 |
| |
|
| Description |
Control_ISC_2
|
| Data processing |
Alignment: ChIP-seq:bowtie. RNA-seq: Tophat, version 2.0.9
ChIP-Seq - peak calling: MACS, version 1.4.2, considering input and local background, p<0.0001 cutoff.
RNA-Seq - normalized and differential expression: Cufflinks, version 2.1.1; settings: -b, -u, and default; Cuffmerge was used with custom 3' 1000 bp reference gtf and default settings; Cuffnorm was used with default settings; Cuffdiff was used with -b, -u and default settings.
Genome_build: mm9
Supplementary_files_format_and_content: ChIP-Seq: Bed file for significant peaks, bigwig file for visualizing aligned sequence tags
Supplementary_files_format_and_content: RNA-Seq: There are two processed data matrices that apply to all the samples. First, is the normalized expression table, prepared using Cuffnorm, which contains the normalized Log2(FPKM+1) expression values for all 9 RNA-seq samples. The second is the differential expression table, prepared using Cuffdiff with an FDR of 5%, which contains average FPKM values for villus, control ISC and Cdx2-del ISC, as well as fold change in expression, P-values, and Q-values corrected for multiple hypothesis testing. The third is the custom gtf used in cuffdiff for correction of 3' bias in the RNA-seq samples.
|
| |
|
| Submission date |
Jul 10, 2015 |
| Last update date |
May 15, 2019 |
| Contact name |
Ramesh Shivdasani |
| E-mail(s) |
ramesh_shivdasani@dfci.harvard.edu
|
| Organization name |
Dana Farber Cancer Institute
|
| Department |
Medical Oncology
|
| Lab |
Shivdasani
|
| Street address |
450 Brookline Ave. Dana 720D
|
| City |
Boston |
| State/province |
MA |
| ZIP/Postal code |
02215 |
| Country |
USA |
| |
|
| Platform ID |
GPL13112 |
| Series (1) |
| GSE70766 |
Distinct processes and transcriptional targets underlie CDX2 requirements in intestinal stem cells and differentiated villus cells |
|
| Relations |
| BioSample |
SAMN03854481 |
| SRA |
SRX1091856 |
