|
| Status |
Public on Dec 30, 2012 |
| Title |
RNA-seq of mES total RNA after Ell3 shRNA replicate 1 |
| Sample type |
SRA |
| |
|
| Source name |
murine embryonic stem cells
|
| Organism |
Mus musculus |
| Characteristics |
treatment: Ell3 shRNA
strain: C57BL/6 (female) x 129/S (male)
cell type: mES
|
| Treatment protocol |
Mouse Ell3 (RMM4534-NM_145973) shRNA constructs were purchased from Open Biosystems. Lentiviral particle preparation and infection were performed as previously described (Lin et al. 2011). Briefly, around 70% confluent 293T cells in a 150 mm tissue culture plate were co-transfected with 8μg of the shRNA construct or a Non-targeting control shRNA, 6 μg of PsPAX2 packaging plasmids and 2 μg of pMD2.G envelope plasmids using FuGENE 6 (Roche 04709691001) or X-tremeGENE 9 (Roche 06365787001). The media was replaced with fresh DMEM supplemented with 10% FBS after 16 hours of transfection. The lentiviral supernatants were collected 48 and 72 hours after the transfection, filtered through 0.45 μm filters and concentrated at 18K rpm for 2 hours. The V6.5 ES cells were infected with concentrated lentiviral particles with polybrene at the concentration of 8 μg/ml. At 24 hours after infection, the ES cells were subjected to selection with 2 ug/ml of puromycin for an additional 48 hours.
|
| Growth protocol |
Mouse embryonic stem cells (KH2 and V6.5) were cultured on irradiated mouse embryonic fibroblast (MEF) feeder layers in 0.1% gelatin-coated tissue culture flask. Cells were grown in DMEM (D6546, Sigma) supplemented with 15% ES-certified fetal bovine serum, 2 mM L-glutamine, 0.1 mM nonessential amino acids, 0.1 mM β-mercaptoethanol, and ecombinant LIF. For ChIP and RNA analysis, cells were grown for one passage off feeders on tissue culture plates for 30 minutes.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
ES cells (V6.5) were infected with lentivirus carrying either Non-targeting shRNA or Ell3 shRNA in the presence of 8 ug/ml of polybrene. 24 hours later, ES cells were selected with 2 ug/ml of puromycin for additional 48 hours and then were grown one passage off feeders for 30 min before harvesting. Total RNA was isolated with the RNeasy (74106 Qiagen) kit, treated with DNase I (M0303L NEB), and re-purified with RNeasy. For total RNA-seq analyses, 2.5 µg of total RNA was depleted of ribosomal RNA with the Ribo-Zero kit from Epicentre (#RZH1046). Ribosomal RNA-depleted samples were used for library preparation with the TruSeq RNA Sample Prep Kit (FC-122-1001 Illumina) for next-generation sequencing.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2000 |
| |
|
| Data processing |
Base-calling was done with Casava v1.8 using default settings for quality filtering.
Alignment of fastq files was done with Bowtie aligner v0.12.7 allowing uniquely mapping reads only and allowing up to two mismatches for ChIP-seq samples. RNA-seq samples were aligned using TopHat v1.4.1 and the Ensembl 63 (ES) and Ensembl 65 (EB5) GTF file allowing unique matches only.
Peak-calling was done with MACS v1.4.1 and filtered at FDR < 5 (0.05)
Genome_build: UCSC mm9
Supplementary_files_format_and_content: Aligned files are BED converted from BAM from bowtie alignments. Peak files are MACS tab-delimited text files, filtered by FDR < 5 (0.05).
|
| |
|
| Submission date |
May 22, 2012 |
| Last update date |
May 15, 2019 |
| Contact name |
Alexander (Garrett) Garruss |
| E-mail(s) |
[email protected]
|
| Organization name |
Stowers Institute for Medical Research
|
| Lab |
Shilatifard
|
| Street address |
1000 East 50th Street
|
| City |
Kansas CIty |
| State/province |
Missouri |
| ZIP/Postal code |
64110 |
| Country |
USA |
| |
|
| Platform ID |
GPL13112 |
| Series (1) |
| GSE38148 |
The RNA Pol II Elongation Factor Ell3 Marks Enhancers in ES Cells and Primes Future Gene Activation |
|
| Relations |
| SRA |
SRX149009 |
| BioSample |
SAMN00997727 |
