|
| Status |
Public on Oct 31, 2018 |
| Title |
Wild type whole kidney rep4 |
| Sample type |
SRA |
| |
|
| Source name |
Mouse embryonic kidney
|
| Organism |
Mus musculus |
| Characteristics |
genotype: Wnt11+/+
tissue: Whole kidney
Stage: E15.5
|
| Treatment protocol |
To isolate nephron progenitors from E15.5 kidneys, GFP+ (Six2TGCtg+ kidneys) were identified by widefield fluorescence microscopy. Each GFP+ kidney pair were isolated, put into a 1.5ml tube, and processed individually. Kidneys were dissociated with collagenase/dispase (Roche, 10269638001) at 37oC for approximately 10-15 minutes, resuspended in PBS containing 2% FBS and 10mM EDTA, and filtered through 40 μm cell strainer (BD Falcon). GFP+ cells were isolated by sorting with a BD FACSAria II. Cells were spun down and resuspended in buffer RLT from the Qiagen RNeasy micro kit.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was isolated from FACS isolated cells or whole kidney using the Qiagen RNeasy micro (cells) or mini (whole kidney) kit following the manufacturer’s instructions. Cells were resuspended and vortexed in buffer RLT and whole kidneys homogenized in buffer RLT using a motorized pestle. Whole kidneys were additionally centrifuged through a QIAshredder to shear genomic DNA and reduce viscosity. The lysates were centrifuged through the provided column to isolate RNA, and the columns subsequently washed, DNase treated, washed, and eluted with RNase-free H2O.
Libraries were constructed from 20-50ng of total RNA by the USC Epigenome Center with the Illumina TruSeq RNA Library Prep Kit V2 following the manufacturer’s instructions with following modifications: End-IT repair from Epicentre was used instead of TruSeq end repair (more favorable volumes). Half of adapter-ligation was amplified for a variable number of cycles depending on input amounts. KAPA Biosystems PCR Master Mix was utilized in place of Illumina's.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2000 |
| |
|
| Description |
Wnt11_WK_RNAseq_DiffExp.txt
WT4 (RPKM)
|
| Data processing |
Sequence files were aligned using TopHat v2.0.8b and Bowtie 2.1.0.0 by the USC Epigenome Center using default parameters.
BAM files were processed to obtain RPKM values using the Partek Genomics Suite 6.6 software.
The Partek ANOVA in Partek Genomics Suite 6.6 software was utilized to find the differential expression of genes ('Differential expression analysis' function) between the two genotypes.
Genome_build: mm9
Supplementary_files_format_and_content: Tab-delimited text files contain gene identifiers, reads, RPKM, and differential expression metrics between wild type and mutant samples
|
| |
|
| Submission date |
Aug 09, 2018 |
| Last update date |
Oct 31, 2018 |
| Contact name |
Lori O'Brien |
| Organization name |
University of North Carolina at Chapel Hill
|
| Department |
Cell Biology and Physiology
|
| Street address |
111 Mason Farm Road
|
| City |
Chapel Hill |
| State/province |
North Carolina |
| ZIP/Postal code |
27599 |
| Country |
USA |
| |
|
| Platform ID |
GPL13112 |
| Series (1) |
| GSE118334 |
Differential gene expression between wild type and Wnt11 mutant embryonic kidneys |
|
| Relations |
| BioSample |
SAMN09786800 |
| SRA |
SRX4523953 |
