|
| Status |
Public on Apr 08, 2015 |
| Title |
Control vector infected DN2 cells #1 |
| Sample type |
SRA |
| |
|
| Source name |
fetal liver progenitor derived DN2 cells
|
| Organism |
Mus musculus |
| Characteristics |
strain: (B6.Cg-Tg(BCL2)25Wehi/J
cell type: In-vitro differentiated DN2 cells
retroviral transduction: Retroviral vector backbone without insert
|
| Treatment protocol |
Fetal liver derived DN cells were harvested from OP9-DL1 co-cultures and infected with retroviruses carrying the indicated constructs. Cells were returned to fresh OP9-DL1 monolayers after 4 hours of infection and GFP+ c-Kit+ CD44+ CD25+ DN2 cells were sorted after ~ 18 hours of culture.
|
| Growth protocol |
Fetal liver progenitors from e14.5 mouse embryos were cultured on OP9-DL1 stromal cells with IL-7 and Flt3L (5 ng/ml each) for 4 days to obtain early DN stage cells.
|
| Extracted molecule |
polyA RNA |
| Extraction protocol |
Sorted cells were lysed in the TRIzol reagent (Invitrogen) and total RNA was extracted as per the manufacturer’s protocol. polyA+ mRNA was isolated using the Dynabeads mRNA purification kit (Invitrogen) and used to generate RNAseq libraries using the NEBNeXT Ultra RNA library preparation kit (E7530) reagents (New England Biolabs) following standard procedures.
polyA+ mRNA was isolated by two rounds of selection with Dynabeads (Invitrogen), fragmented to an average size of 200 bp by Mg2+ mediated hydrolysis and used to synthesize cDNA by random priming. The cDNA was end-repaired, the blunt ends were phosphorylated and Illumina adapters were ligated to the fragments. ~300 bp cDNA fragments were then purified by gel electrophoresis and SPRI (Agencourt AMPure XP catalog #A63880) beads were used to remove shorter fragments (<100 bp). The cDNA was then PCR amplified for 15 cycles and the resulting DNA fragments were purified once again with SPRI beads and sequenced on the Illumina HiSeq 2000 sequencer following the manufacturer's protocol.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2000 |
| |
|
| Description |
Empty vector control - 1
|
| Data processing |
Sequence reads from each sample were trimmed to remove the adapter sequences and the resulting 50 bp reads were mapped to the NCBI37/mm9 mouse genome assembly
Alignment was done using Tophat (v.2.0.6). command: tophat --no-novel-juncs --library-type fr-unstranded -G Mus_musculus.NCBIM37.66.gtf
Abundance measurements were done using Cufflinks (v.2.1.1). cufflinks (fpkm) command: cuffdiff --dispersion-method blind --library-type fr-unstranded --no-effective-length-correction --library-norm-method quartile -u -b mm9.fa --emit-count-tables Mus_musculus.NCBIM37.66.gtf
Genome_build: UCSC mm9, NCBI37
Supplementary_files_format_and_content: Tab-delimited files with fpkm values generated from Cufflinks
|
| |
|
| Submission date |
Jan 27, 2015 |
| Last update date |
May 15, 2019 |
| Contact name |
Ameya Champhekar |
| E-mail(s) |
[email protected]
|
| Organization name |
University of California, Los Angeles
|
| Department |
Medicine, HemOnc Division
|
| Street address |
700 Tiverton Ave, 11-272 Factor Bldg
|
| City |
Los Angeles |
| State/province |
California |
| ZIP/Postal code |
90095-1678 |
| Country |
USA |
| |
|
| Platform ID |
GPL13112 |
| Series (1) |
| GSE65344 |
PU.1 regulates T-lineage gene expression and progression via indirect repression during early T-cell development |
|
| Relations |
| BioSample |
SAMN03295402 |
| SRA |
SRX853954 |
