GEO Logo
   NCBI > GEO > Accession DisplayHelp Not logged in | LoginHelp
GEO help: Mouse over screen elements for information.
Sample GSM976933 Query DataSets for GSM976933
Status Public on Dec 06, 2012
Title KD5day-plusDox3 (GAGTACG)
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: GAGTACG
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 ( . 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) (
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
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
SRA SRX171017
BioSample SAMN01095615

Supplementary file Size Download File type/resource
GSM976933_KDplusDox3_GAGTACG_vs_KDnoDox_GTATTTG_all.txt.gz 790.0 Kb (ftp)(http) TXT
GSM976933_KDplusDox3_GAGTACG_vs_KDnoDox_GTATTTG_significant.txt.gz 30.4 Kb (ftp)(http) TXT
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

| NLM | NIH | GEO Help | Disclaimer | Accessibility |
NCBI Home NCBI Search NCBI SiteMap