|
| Status |
Public on Nov 13, 2014 |
| Title |
Cre-infected cKO MEFs H3K27me3 ChIPseq biological replicate 3 |
| 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
chip antibody: H3K27me3
chip vendor/catalog#: Millipore #07-449
|
| 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 |
genomic DNA |
| 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).
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| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| 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 |
| E-mail(s) |
julsage@stanford.edu
|
| 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 |
|
| Relations |
| SRA |
SRX183340 |
| BioSample |
SAMN01162191 |
