|
| Status |
Public on Feb 17, 2012 |
| Title |
Lin28-RV GFP- DP thymocytes |
| Sample type |
SRA |
| |
|
| Source name |
Lin28-RV GFP- DP thymocytes
|
| Organism |
Mus musculus |
| Characteristics |
strain: C57BL/6
cell type: thymocytes
variation: CD3- CD4+ CD8+ DP
|
| Treatment protocol |
Six weeks post-adoptive transfer, RNA was extracted from FACS-sorted GFP+ and GFP- DP thymocytes (CD4+ CD8+ CD3-) and pooled from three retrogenic bone marrow chimeric mice. RNA concentration was determined using the Qubit RNA broad range assay and fluorometer (Invitrogen). To confirm the concentration and determine the integrity of the RNA, we used the Eukaryote Total RNA nano Series II chip on a 2100 Bioanalyzer (Agilent).
|
| Growth protocol |
Retrogenic bone marrow chimeras were generated by transduction of wildtype bone marrow hematopoietic stem and progenitor cells with a retrovirus encoding Lin28a in vitro in the presence of SCF (50 ng/ml), IL-6 (10 ng/ml), IL-3 (5ng/ml), FLT-3L (5ng/ml) and IL- 7 (5ng/ml). A mixture of transduced and untransduced cells were injected into sublethally (450RADS) irradiated Rag1-/- hosts at 2-4 million cells per host.
|
| Extracted molecule |
polyA RNA |
| Extraction protocol |
RNAseq library preparation was performed starting with 3.3 μg of total RNA using the Truseq RNA sample prep kit (Illumina) following manufacturer’s protocol. Briefly, oligo-dT purified and fragmented mRNA was subjected to first and second strand cDNA synthesis. cDNA fragments were blunt-ended, ligated to Illumina adaptors, and PCR amplified to enrich for the fragments ligated to adaptors. The resulting cDNA libraries were verified and quantified on Agilent Bioanalyzer, and sequenced by paired-end RNA-seq using the GAIIx sequencing system (Illumina).
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina Genome Analyzer IIx |
| |
|
| Data processing |
DP_Lin28RV_GFP_KO.bedgraph; genome build: mm9
Raw reads were mapped to the mouse genome (NCBI 37, mm9) using the spliced read aligner TopHat version 1.3.1 with option --mate-inner-dist 160 --coverage-search -- microexon-search --max-multihits 20. bamToBed (BedTools v2.11.0, http://code.google.com/p/bedtools/) was used to convert resulting bam files to bed files, then genomeCoverageBed (BedTools v2.11.0, http://code.google.com/p/bedtools/) was used to convert bed files to bedgraph files.
|
| |
|
| Submission date |
Jan 04, 2012 |
| Last update date |
May 15, 2019 |
| Contact name |
Cuong Kieu Nguyen |
| E-mail(s) |
nguyenck@mail.nih.gov
|
| Phone |
301-451-3227
|
| Organization name |
NIH
|
| Department |
NIAID
|
| Lab |
Laboratory of Immunology
|
| Street address |
9000 Rockville Pike
|
| City |
Bethesda |
| State/province |
MD |
| ZIP/Postal code |
20892 |
| Country |
USA |
| |
|
| Platform ID |
GPL11002 |
| Series (1) |
| GSE34854 |
Transcriptome of double-positive thymocyte precursors transduced with a retrovirus encoding Lin28a |
|
| Relations |
| Reanalyzed by |
GSE80797 |
| SRA |
SRX113964 |
| BioSample |
SAMN00769652 |
