|
| Status |
Public on Oct 28, 2020 |
| Title |
Group3-Asc-TMDKOAsc-3 |
| Sample type |
SRA |
| |
|
| Source name |
Ascending colon
|
| Organism |
Mus musculus |
| Characteristics |
strain: Mixed BL/6-129SvJ
tissue: Ascending colon
genotype: TM-DKOAsc
cage: C
Sex: F
age: 3.2 months
|
| Treatment protocol |
To generate mice with induced O-glycan deficiency in either the ascending or descending colon, mutant or WT littermates were injected intraperitoneally with 0.5 mg tamoxifen (TM, or ~20 mg/kg, MP Bio-medicals) in an ethanol/sunflower seed oil mixture (1:9 (v/v)) daily for 5 consecutive days, and mice were analyzed for deletion 5 or 10 days post final TM treatment. The dosage of TM was determined after serial titrations and characterization of different TM dosages in different mutant models.
|
| Growth protocol |
To produce mice with induced deficiency of O-glycans specifically in colon, C1galt1-floxed (C1galt1f/f);C3GnT-/- females were crossed with previously generated C1galt1f/f;VillinCre-ERT2 (TM-IEC C1galt1f/f) mice on a C3GnT+/- background to generate C1galt1f/f;VillinCre-ERT2;C3GnT-/- (TM-DKO) and WT littermates. All mutant mice and WT littermates were on a C57BL/6J (BL/6) and 129/SvImJ (129/Sv) mixed background. Inbred WT mice (BL/6, Balb/c, and 129SvJ mice) were obtained from the Jackson Laboratory. Outbred WT CD1 mice and Swiss Webster (SW) mice and Sprague Dawley rat were bred in house. Animals were fed standard chow (PicoLab Rodent Diet 20; LabDiet 5053). Animals were housed in a specific pathogen free facility (SPF). All animal experiments were approved by the Institutional Animal Care and Use Committee of Oklahoma Medical Research Foundation.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was extracted using the Zymo Direct-Zol RNA MiniPrep Plus (cat# R2070, Irvine, CA) as per the manufacturer’s instructions. Total RNA was quantified and 260/280 ratios determined using Nanodrop (all samples with a 260/280>1.97). 1μg of total RNA was used as starting material for each sample.
RNA sequencing and library preparation was performed by the UCLA Technology Center for Genomics and Bioinformatics with Kapa hyper Prep Kits with RiboErase. The workflow consists of rRNA depletion, cDNA generation, and end repair to generate blunt ends, A-tailing, adaptor ligation and PCR amplification. Different adaptors were used for multiplexing samples in one lane. Sequencing was performed on Illumina Hiseq3000 for a single read 50 run.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 3000 |
| |
|
| Description |
Pro-Group3-KO_3
|
| Data processing |
Data quality check was done on an Illumina SAV. Demultiplexing was performed with the Illumina Bcl2fastq2 v 2.17 program.
The STAR ultrafast universal RNA-seq aligner (v2.7) was used to generate the genome index and perform single-ended alignments. Reads were aligned to a genome index that includes both the genome sequence (GRCm38 primary assembly) and the exon/intron structure of known gene models (Gencode M20 genome annotation). Alignment files were used to generate strand-specific, gene-level count summaries with STAR's built-in gene counter. Differential expression analysis was performed with DESeq2.
Data in supplementary file ProDisExpression.genesfpkm.txt was generated from gene-level count summaries after per-sample normalization in units of FPKMs: counts for kilobase of gene mappable length and million of total counts.
Genome_build: GRCm38/mm10
Supplementary_files_format_and_content: Tab delimited, per-sample, gene expression estimates in units of FPKMs
|
| |
|
| Submission date |
Jun 21, 2019 |
| Last update date |
Oct 28, 2020 |
| Contact name |
David Casero |
| E-mail(s) |
dcasero@ucla.edu
|
| Phone |
3108250274
|
| Organization name |
UCLA
|
| Department |
Pathology and Laboratory Medicine
|
| Street address |
610 Charles Young Drive East
|
| City |
Los Angeles |
| State/province |
CA |
| ZIP/Postal code |
90095 |
| Country |
USA |
| |
|
| Platform ID |
GPL21493 |
| Series (2) |
| GSE133174 |
Ascending colon-derived mucin-type O-glycans form key mucus layers encapsulating microbiota in the colon (RNA-seq) |
| GSE133257 |
Ascending colon-derived mucin-type O-glycans form key mucus layers encapsulating microbiota in the colon |
|
| Relations |
| BioSample |
SAMN12108701 |
| SRA |
SRX6183229 |
