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Sample GSM997575 Query DataSets for GSM997575
Status Public on Nov 13, 2014
Title Cre-infected cKO MEFs RNAseq biological replicate 2
Sample type SRA
Source name conditional KO Rb MEF
Organism Mus musculus
Characteristics cell type: MEFs
strain: mixed B6;129 background
gfp/cre-infected: Cre
4f: no 4F
Treatment protocol To conditionally delete Rb cells were infected with titered Adeno-Cre or Adeno-GFP as a control, 1x10^6 cells at a time. In the RNAseq or Rb ChIPseq samples, the cells were then infected with a puro-selectable 4F construct or an empty pSicoR-Puro. Puro was added the following day and selection was maintained until the third day when the cells were harvested for sample processing. For the histone ChIPseq, the MEFs were infected with Adeno_GFP or Adeno-Cre and then expanded no more than 3 passages to opbtain enough cells.
Growth protocol Cells were grown at 37oC in 5% CO2 in standard media (DMEM, 10% serum, glutamine, penn/strep)
Extracted molecule polyA RNA
Extraction protocol Purified RNA was isolated using Trizol and a Qiagen RNEasy Kit. After assessing RNA quality using an Agilent 2100 Bioanalyzer , cDNA was made using an Ovation® RNA-Seq System V2 (NuGEN) and sonicated using a Covaris S2 Sonicator. Sequencing libraries were generated using the NEBNext® DNA Sample Prep Master Mix Set (New England Biolabs) and purified using Agencourt® Ampure® XP beads (Bechman Coulter).
Library strategy RNA-Seq
Library source transcriptomic
Library selection cDNA
Instrument model Illumina HiSeq 2000
Data processing RNAseq: The reads were mapped using Tophat (v2.0.3) calling Bowtie (v2.0.0.6) and using samtools (v0.1.18.0) to the NCBIM37 genome annotation. Cufflinks (v2.0.1) was then run with the -G option to the annotated NCBIM37 genes and a mask file containing rRNA, Mt_rRNA, and Mt_tRNA sequences. The assemblies were compiled using Cuffmerge with the -g option to annotated genes and -s to the NCBIM37 genome. Cuffdiff was then run using the mask file, the --frag-bias-correct option, and performing upper quartile normilization (--upper-quartile-norm) to more accuratley call low expressed genes.
RB ChIPseq: The reads were mapped to the NCBIM37 genome using BWA. Aligned reads were converted to bed files using Samtools (v0.1.18) and bedTools (v2.16.2). Bed files were converted to aln files using CisGenome (v2.0) and then the "Two Sample Peak Calling" was run with both biological replicates and their respective 10% Total Input samples with the following parameters modified (E=450, B=25, W=2, and without the standardization of win statistics). Output bar files were converted to wig.
Histone ChIPseq: The reads were mapped to the NCBIM37 genome using BWA and peaks called using MACS (v1.4.1) without model building (--nomodel) and the band width set to the average library length calculated using a bioanalyzer.
Submission date Sep 04, 2012
Last update date May 15, 2019
Contact name Julien Sage
Phone (650) 723-5113
Organization name Stanford University
Department Pediatrics
Lab Sage
Street address 265 Campus Drive, SIM1 G2078
City Stanford
State/province CA
ZIP/Postal code 94305-5457
Country USA
Platform ID GPL13112
Series (1)
GSE40594 Localization of the Rb tumor suppressor in MEFs during reprogramming to iPS and the consequence of Rb loss on the transcriptional profile and histone landscape in these cells
Reanalyzed by GSE80797
SRA SRX183325
BioSample SAMN01162176

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

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