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Sample GSM856150 Query DataSets for GSM856150
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_WT.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, was used to convert resulting bam files to bed files, then genomeCoverageBed (BedTools v2.11.0, 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
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
Reanalyzed by GSE80797
SRA SRX113963
BioSample SAMN00769651

Supplementary file Size Download File type/resource
GSM856150_DP_Lin28RV_GFP_WT.bedgraph.gz 270.8 Mb (ftp)(http) BEDGRAPH
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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