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Sample GSM2252944 Query DataSets for GSM2252944
Status Public on Oct 01, 2016
Title 0 hour - Replicate 1
Sample type SRA
 
Source name Lys-GFP-ER-HoxA9 cells
Organism Mus musculus
Characteristics strain: C57Bl/6
Treatment protocol Culture out of beta-estradiol for the indicated times
Growth protocol RPMI1640 + 10% FBS + Pen/Strep + Glutamine + Stem Cell Factor + Beta-estradiol (0.5 micromolar)
Extracted molecule total RNA
Extraction protocol Qiagen RNEasy PLUS kit with additional on-column DNAse step
Total RNA is quantified using the Quant-iT™ RiboGreen® RNA Assay Kit and normalized to 5ng/ul. An aliquot of 200ng for each sample is transferred into library preparation which is an automated variant of the Illumina TruSeq™ Stranded mRNA Sample Preparation Kit. This method preserves strand orientation of the RNA transcript. It uses oligo dT beads to select mRNA from the total RNA sample. It is followed by heat fragmentation and cDNA synthesis from the RNA template. The resultant 500bp cDNA then goes through library preparation (end repair, base ‘A’ addition, adapter ligation, and enrichment) using Broad designed indexed adapters substituted in for multiplexing. After enrichment the libraries were quantified with qPCR using the KAPA Library Quantification Kit for Illumina Sequencing Platforms and then pooled equimolarly. The entire process is in 96-well format and all pipetting is done by either Agilent Bravo or Hamilton Starlet.
Pooled libraries are normalized to 2nM and denatured using 0.1 N NaOH prior to sequencing. Flowcell cluster amplification and sequencing were performed according to the manufacturer’s protocols using either the HiSeq 2000 or HiSeq 2500. Each run is a 101bp paired-end with an eight-base index barcode read. Data is analyzed using the Broad Picard Pipeline which includes de-multiplexing and data aggregation.
 
Library strategy RNA-Seq
Library source transcriptomic
Library selection cDNA
Instrument model Illumina HiSeq 2000
 
Data processing Base calls performed with Illumina RTA (version unknown)
Illumina base calls converted to BAM with Picard IlluminaBasecallsToSam version 1.764
RNA-Seq reads were aligned to the mm10 genome assembly, including the sequence of eGFP, and counted with STAR version 2.4.2a with default settings
Genome_build: mm10
Supplementary_files_format_and_content: a tab-delimited text file includes raw read counts for each sample
 
Submission date Jul 26, 2016
Last update date May 15, 2019
Contact name David B Sykes
E-mail(s) [email protected]
Organization name Massachusetts General Hospital
Department Center for Regenerative Medicine
Street address 185 Cambridge St
City Boston
State/province Massachusetts
ZIP/Postal code 02114
Country USA
 
Platform ID GPL13112
Series (1)
GSE84874 Time course of myeloid differentiation in the Lysozyme-GFP ER-HoxA9 cells following estradiol withdrawal
Relations
BioSample SAMN05441447
SRA SRX1978410

Supplementary data files not provided
SRA Run SelectorHelp
Raw data are available in SRA
Processed data are available on Series record

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