|
| Status |
Public on Sep 21, 2018 |
| Title |
Anti-NRR antibody-treated DN3-2 |
| Sample type |
SRA |
| |
|
| Source name |
Anti-NRR antibody-treated
|
| Organism |
Mus musculus |
| Characteristics |
strain: C57BL/6
cell type: DN3 thymocytes
timing: 24 hours post antibody injection
|
| Treatment protocol |
Rosa26CreERT2Zmiz1f/f (DeltaTamCre) mice and Rosa26CreERT2 (TamCre) control mice (4 mice per group) were injected with 0.2 mg/g tamoxifen IP. DN3 cells were sorted at 43-44 hours post-injection. MxCreZmiz1f/f mice and control MxCre mice (3 mice per group) were injected with 40 mcg pI-pC IP every 2 days for 5 times. DN3 cells were sorted at 8 weeks post-injection. Mice were injected with 10 mg/kg anti-NRR Notch1 antibody (Genentech) or the isotype control IgG antibody (Genentech) IP prior to sorting of DN3 cells at 24 hours post-injection.
|
| Growth protocol |
Dapi-CD44+CD25-Lineage- DN3 thymocytes were sorted with a BD AriaII from live mice directly into RLT-plus buffer (Qiagen)
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted from 100K-400K DN3 cells using the RNeasy Plus Micro kit (Qiagen) according to the manufacturer’s protocol.
DeltaTamCre and TamCre sequencing libraries were prepped from 100 ng of total RNA using the Illumina TruSeq mRNA Sample Prep v2 kit (RS-122-2001, RS-122-2002) accrording to manufacturer's protocol by the University of Michigan Sequencing Core. DeltaMxCre, MxCre, Anti-NRR antibody, and Control IgG antibody sequencing libraries were prepped from 50 ng of total RNA using the Clontech SMARTer Ultra low input RNA kit (6348493) accrording to manufacturer's protocol by the University of Michigan Sequencing Core.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2500 |
| |
|
| Description |
Sample_51576
|
| Data processing |
FastQC version 0.10.0 (http://www.bioinformatics.bbsrc.ac.uk/projects/fastqc/) was used to identify features of the raw data that may indicate quality problems
The Tuxedo Suite was used to align reads to the reference transcriptome (UCSC mm10) (http://genome.ucsc.edu/) using TopHat (version 2.0.9) and Bowtie (version 2.1.0.0). Default parameter settings were used for alignment, with the exception of: “--b2-very-sensitive” telling the software to spend extra time searching for valid alignments, as well as “--no-coverage-search” and “--no-novel-juncs” to limit the read mapping to known transcripts.
FastQC was used for a second round of quality control (post-alignment), to ensure that only high quality data would be input to expression quantitation and differential expression analysis
Cufflinks/CuffDiff (version 2.1.1) was used for expression quantitation and differential expression analysis, using UCSC mm10.fa as the reference genome sequence and UCSC mm10.gtf as the reference transcriptome annotation. Thsi analysis used parameter settings: “--multi-read-correct” to adjust expression calculations for reads that map in more than one locus, as well as “--compatible-hits-norm” and “--upper-quartile –norm” for normalization of expression values.
Locally developed scripts were used to format and annotate the differential expression data output from CuffDiff. Briefly, we identified genes and transcripts as being differentially expressed based on three criteria: test status = “OK”, FDR < 0.05 and fold change ≥ 1.5. Genes and isoforms were annotated with NCBI Entrez GeneIDs, text description, and Gene Ontology (GO) (http://david.abcc.ncifcrf.gov/) terms using NCBI annotation.
Genome_build: mm10
Supplementary_files_format_and_content: Table of raw reads counts in .txt format
|
| |
|
| Submission date |
Nov 08, 2017 |
| Last update date |
May 15, 2019 |
| Contact name |
Richard C McEachin |
| E-mail(s) |
mceachin@umich.edu
|
| Organization name |
University of Michigan
|
| Department |
DCM&B
|
| Street address |
2800 Plymouth Road
|
| City |
Ann Arbor |
| State/province |
United States |
| ZIP/Postal code |
48109 |
| Country |
USA |
| |
|
| Platform ID |
GPL17021 |
| Series (2) |
| GSE106683 |
The Zmiz1-Notch1 interaction induces Myc expression to drive steady state and stress thymopoiesis [DN3] |
| GSE116125 |
The Zmiz1-Notch1 interaction induces Myc expression to drive steady state and stress thymopoiesis |
|
| Relations |
| BioSample |
SAMN08000617 |
| SRA |
SRX3375237 |
