|
| Status |
Public on Apr 16, 2015 |
| Title |
RNA-seq of PolyA Minus RNA from Mouse ES cells with Floxed ATRX Allele |
| Sample type |
SRA |
| |
|
| Source name |
ES cells
|
| Organism |
Mus musculus |
| Characteristics |
strain: Cast x 129
sample type: Strand Specific Library Preparation
treatment: AdCre
|
| Treatment protocol |
Mouse ES cells were generated from a male Castaneus x female 129 (ATRX Flox) cross. Castx129 ATRX Flox ES cells were transduced with either an Adenoviral GFP or Adenoviral Cre-recombinase GFP cassette. Cells were sorted in GFP positive fractions at 48 hours post transduction. RNA was extracted at 5 days post-transduction
|
| Growth protocol |
Mouse ES cells were cultured according to standard protocols.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was isolated with Tri-Reagent (Sigma). The RNA quality was assessed using Agilent Bioanalyser. Only RNA with overall RIN score >9 was used for further procedures. Subsequently, RNA was treated with DNAseI (DNA-free, Ambion) according to the manufacturer’s protocol kit according to the protocol. Good quality and DNA-free RNA was subjected to polyA fractionation using PolyA selection kit (Promega) using the standard kit protocol. The RNA fraction was precipitated overnight and resuspended with water. The quality of obtained RNA samples was assessed using PicoChip (Agilent). A Paired sequencing library was generated using the standard Illumina prtocol.
Prior to library prep, the polyA- samples were treated with EpiCentre's Ribo-Zero™ Magnetic Kit (Human/Mouse/Rat). All libraries were prepped with the following kit. NEBNext® Ultra™ Directional RNA Library Prep Kit for Illumina®. The protocol was modified to make use of our own custom 8bp indexes (Lamble et al. 2013). The prepared libraries then sequenced on a HiSeq2500 in rapid mode (no version number as original chemistry), 50bp paired end.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2500 |
| |
|
| Description |
PolyA- RNA
An adenoviral packaged Cre-recombinase cassette with a GFP marker (Vector Biolabs 1779) (AdCre) was used to generate ATRX KO ES cells.
|
| Data processing |
Reads were mapped to the mm9 mouse genome build using TopHat 1.1.4b
Read coverage was calculated genome-wide to produce strand specific UCSC Wig files
Genome_build: mm9
Supplementary_files_format_and_content: Strand specific UCSC Wig files of read coverage from tophat output.
|
| |
|
| Submission date |
Jul 09, 2014 |
| Last update date |
May 15, 2019 |
| Contact name |
Jim Hughes |
| E-mail(s) |
jim.hughes@imm.ox.ac.uk
|
| Phone |
1865222113
|
| Organization name |
University of Oxford
|
| Department |
MHU
|
| Lab |
Genome Biology Group
|
| Street address |
Weatherall Institute Of Molecular Me
|
| City |
oxford |
| ZIP/Postal code |
OX3 9DS |
| Country |
United Kingdom |
| |
|
| Platform ID |
GPL17021 |
| Series (2) |
| GSE59269 |
Transcriptome analysis of WT and ATRX KO Cast x 129 mouse ES cells |
| GSE59271 |
WT and ATRX KO |
|
| Relations |
| BioSample |
SAMN02905725 |
| SRA |
SRX648563 |
