|
| Status |
Public on May 03, 2012 |
| Title |
Cytoplasmic_30min |
| Sample type |
SRA |
| |
|
| Source name |
Macrophage
|
| Organism |
Mus musculus |
| Characteristics |
strain: C57BL/6
subcellular fraction: Cytoplasmic
stimulation time: 30 minutes
stimulation type: Lipid A
|
| Treatment protocol |
Lipid A was added at a concentration of 100ng/ml to each 25 cm plate for the times indicated.
|
| Growth protocol |
Bone marrow from mixed sex mice were plated at a density of 7x10^5 cells/ml into DMEM containing 20% FBS supplemented with sodium pyruvate, L-glutamine and M-CSF. Media was changed on day three of differentiation. Adherent cells were scraped and replated at a density of 1x10^6 cells/mL into 15 cm plates on day five of differentiation. Cells were stimulated on day six.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Cells were fractionated according to the protocol described in Pandya-Jones and Black, RNA 2009. Briefly, cells were lysed in 0.15% NP40 buffer and spun over a sucrose cushion to obtain nuclei. Nuclei were lysed in buffer containing 1% NP40 and 1M urea. RNA from the cytoplasmic and nucleoplasmic lysates as well as the chromatin pellet was purified using Qiagen RNeasy columns according to manufacturer protocol. 10 ug of RNA from each sample was depleted of ribosomal RNA using the Invitrogen Human/Mouse Ribominus kit. Libraries were constructed from 500 ng of rRNA-depleted RNA using the dUTP method described in Levin et al, Nat Methods 2010 was used to generate strand-specific sequencing libraries.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2000 |
| |
|
| Data processing |
BAM: Sequence reads were obtained and mapped to the mouse mm9 genome using Tophat. Uniquely mapping reads with two or fewer mismatches were retained.
|
| |
|
| Submission date |
Oct 12, 2011 |
| Last update date |
Jan 25, 2024 |
| Contact name |
Stephen Smale |
| E-mail(s) |
[email protected]
|
| Organization name |
University of California Los Angeles
|
| Department |
MIMG
|
| Lab |
Smale Lab
|
| Street address |
6-730 MRL 675 Charles E. Young Drive South
|
| City |
Los Angeles |
| State/province |
Ca |
| ZIP/Postal code |
90024 |
| Country |
USA |
| |
|
| Platform ID |
GPL13112 |
| Series (1) |
| GSE32916 |
Analysis of Pro-Inflammatory Gene Activation and RNA Processing by RNAseq of Nascent Transcripts I |
|
| Relations |
| SRA |
SRX100829 |
| BioSample |
SAMN00738686 |
