|
| Status |
Public on Dec 06, 2012 |
| Title |
KD5day-plusDox2 (CTCAATG) |
| Sample type |
SRA |
| |
|
| Source name |
neural progenitors derived from mouse embryonic stem cells, Geminin knockdown
|
| Organism |
Mus musculus |
| Characteristics |
es cell line: A2lox
cell type: embryonic stem cell-derived neural precursors
treatment: plus Dox
library label: CTCAATG
|
| Treatment protocol |
ES cells enabling doxycycline-dependent Geminin knockdown (described previously, Yellajoshyula et al., PNAS 108(8), pg. 329) were treated with 500ng doxycycline from day 3 to day 5 of neuronal differentiation in N2B27 medium. RNA samples were collected for library construction and sequencing on day 5.
|
| Growth protocol |
The A2lox mouse ES cell line and clonal derivatives were routinely propagated on feeder cells (mouse embryonic fibroblast cells; Chemicon) in DMEM (Invitrogen) supplemented with 15% FCS (HyClone), 1 mM sodium pyruvate, 1 mM non-essential amino acids, 1 mM L-glutamine (Invitrogen), 10−4 M 2-mercaptoethanol (Sigma), and 10exp3 units per milliliter of leukemia inhibitory factor (ESGRO; Chemicon). Monolayer neural differentiation of ES cells was done as previously described (Yellajoshyula et al., PNAS 108(8), pg. 329). Briefly, ES cells cultured on feeder cells were dissociated and plated at 1 × 10exp5/cm2 for 1 day on 0.1% gelatin-coated tissue culture dishes. After 24 h, cells were plated onto 0.1% gelatin-coated tissue culture dishes at low density (1 × 10exp4 cells/cm2) in N2B27 medium and cultured for 5 days.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was isolated from the cells using the Trizol Plus RNA purification system (Invitrogen). Library construction was conducted by the Genome Technology Access Center (GTAC) at Washington University School of Medicine, according to standard protocols for Illumina HiSeq NextGen sequencing (http://gtac.wustl.edu/services/sequencing/library-preparation.php) . Four samples indexed with the indicated sequence tags were sequenced on a lane of the Illumina HiSeq.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2000 |
| |
|
| Description |
Total RNA was isolated from GemKD cells differentiated in N2B27 media for 5 days with or without addition of 500 ng Dox for Gem knockdown from day 3. These samples were used for RNA-seq analysis (Illumina HiSeq2000 platform) by the Genome Technology Access Center (GTAC) at Washington University School of Medicine in St. Louis. Three independent experiments were conducted to generate Geminin knockdown (plus Dox) samples and these were compared to the no-Dox control.
|
| Data processing |
Sequence alignment to the mm9 genome was performed by GTAC using TopHat with the following settings: a. min anchor=5; m. splice mismatches=1; i. min intron length=10; I. max intron length=500000; g. max # of multi hits=100 (solexa 1.3 quals)
Transcript abundance was determined by GTAC using Cufflinks with option I=500000.
Genes that were differentially expressed between the experimental (plus Dox) and control (no Dox) samples were defined using Cuffdiff (Trapnell et al., Nature Protocols 7: 562-78) (http://cufflinks.cbcb.umd.edu/manual.html).
Transcripts were considered as differentially expressed upon Gem knockdown if data met statistical significance cutoffs in Cuffdiff (sufficient sequence alignments were obtained for analysis and transcript had significant change in FPKM value (normalized transcript abundance; fragments per kb of exon per million fragments mapped) between the no Dox and plus Dox sample pairs) in at least two of the three independent experiments.
Genome_build: mm9
Supplementary_files_format_and_content: Cuffdiff output: Matrix files from comparisons of experimental versus control samples, which were used to define differentially expressed genes.
|
| |
|
| Submission date |
Jul 25, 2012 |
| Last update date |
May 15, 2019 |
| Contact name |
Kristen L Kroll |
| E-mail(s) |
kkroll@wustl.edu
|
| Organization name |
Washington University School of Medicine
|
| Department |
Developmental Biology
|
| Lab |
Kristen Kroll
|
| Street address |
320 McDonnell Sciences/660 S. Euclid Ave.
|
| City |
Saint Louis |
| State/province |
MO |
| ZIP/Postal code |
63110 |
| Country |
USA |
| |
|
| Platform ID |
GPL13112 |
| Series (2) |
| GSE39657 |
Geminin knockdown in embryonic stem cell-derived neural precursors |
| GSE39673 |
Geminin regulates the transcriptional and epigenetic status of neuronal fate promoting genes during mammalian neurogenesis |
|
| Relations |
| SRA |
SRX171016 |
| BioSample |
SAMN01095614 |
