|
| Status |
Public on Mar 01, 2012 |
| Title |
Ago 2 immunoprecipitated RNA extracted from MCMV infected NIH-3T3 cells |
| Sample type |
SRA |
| |
|
| Source name |
NIH-3T3_MCMV_Ago_IP
|
| Organism |
Mus musculus |
| Characteristics |
cell line: NIH-3T3
cell type: fibroblast
infected with: murine cytomegalovirus (MCMV)
ip antibody: mAgo2-antibody
ip antibody vendor: Wako
ip antibody cat #: 2D4
|
| Treatment protocol |
Ago2-immunoprecipitation was performed as recently described [Dolken et al. Cell Host Microbe 7: 324-334]. In short, 6 µg of purified monoclonal mAgo2-antibody (2D4, Wako) was added to 5 mL of RPMI-medium and incubated with 30 µL of Protein-G-Sepharose beads (GE Healthcare) in Pierce centrifuge columns (Thermo Scientific) under constant rotation at 4 °C over night. Columns were drained by gravity flow and washed once with the lysis buffer. Beads were subsequently incubated with 5 mL of cell lysates for 2.5 h under constant rotation at 4 °C. After incubation, the beads were washed four times with IP wash buffer (300 mM NaCl, 50 mM Tris HCl pH 7.5, 5 mM MgCl2, 0.1% NP-40, 1 mM NaF) and once with PBS to remove residual detergents. RNA was recovered from the beads by adding 700 µl of Qiazol to the columns. After 5 min the Qiazol lysates were collected from the columns. This step was repeated once and the Qiazol lysates were combined. RNA was prepared using the miRNeasy kit (Qiagen) according to the manufacturer's instructions. RNA samples were eluted in 30 µL of H2O.
|
| Growth protocol |
NIH-3T3 fibroblasts (ATCC: CRL-1658) were cultured in DMEM medium containing 5% fetal calf serum and penicillin/streptomycin. For viral infection, NIH-3T3 cells were infected with wild-type MCMV at an MOI of 10.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Libraries were prepared using either total RNA extracted with Trizol from mock-infected NIH-3T3 cells, or from cells infected with MCMV for 6 hours as well as from RNA extracted after Ago2 IP from the same samples. For libraries generated from total RNA, 10 μg of total RNA was used in the initial step. For libraries generated from small RNAs incorporated into Ago2 containing RISC complexes, a fraction of the mAgo2 IPs from either MCMV infected (6hpi), or control cells were used. Samples were size fractionated on a 17.5% PAGE gel, and small RNAs between 19 and 33 nt were excised and cloned as previously described (Pfeffer S, 2007. Identification of virally encoded microRNAs. Methods Enzymol. 427:51-63), except that small RNA PCR products were not concatamerized and instead sent directly for sequencing.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
size fractionation |
| Instrument model |
Illumina Genome Analyzer IIx |
| |
|
| Data processing |
Counts: Raw reads were first processed using the Dustmasker program (Morgulis A et al., 2006. A fast and symmetric DUST implementation to mask low-complexity DNA sequences. J Comput Biol 13:1028-1040) to filter out low complexity reads, and then trimmed using the FASTX-Toolkit (http://hannonlab.cshl.edu/fastx_toolkit) to remove the 3' adaptor. Only reads from 15 to 32 nt were kept for further analysis. Remaining sequences with counts included.
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| |
|
| Submission date |
Dec 15, 2011 |
| Last update date |
May 15, 2019 |
| Contact name |
Sébastien Pfeffer |
| Organization name |
CNRS - Institut de Biologie Moléculaire et Cellulaire (IBMC)
|
| Department |
UPR 9002 - Architecture et Réactivité de l'ARN
|
| Lab |
RNA regulation in viral infections
|
| Street address |
2 allée Konrad Roentgen
|
| City |
Strasbourg |
| ZIP/Postal code |
67084 |
| Country |
France |
| |
|
| Platform ID |
GPL11002 |
| Series (1) |
| GSE34475 |
Degradation of cellular miR-27 by a novel, highly abundant viral transcript is important for efficient virus replication in vivo. |
|
| Relations |
| SRA |
SRX111826 |
| BioSample |
SAMN00765675 |
