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Sample GSM849858 Query DataSets for GSM849858
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.
 
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

Data table header descriptions
SEQUENCE
COUNT

Data table
SEQUENCE COUNT
AAAACGACCCTGTAT 3
AAAAGAGTTGGTACA 1
AAAGCGGGCAGTGAG 2
AAAGCTGGGTTGAGA 1
AAAGGCAGTTCCTGA 14
AAAGGCTGGGGTACC 4
AAATCGCACCCGTCT 3
AAATCTGGGTTGAGA 4
AAATGACTCGGTGCT 1
AACACAGAACTTTGT 1
AACACGTGCGCGTGA 9
AACAGACTGATGTTG 1
AACAGTCCGACGATC 13
AACCACCCTCTAAAC 1
AACCCGTAGATCCGA 1
AACCCTAACTTGTGA 1
AACCGGGGGAACTGA 1
AACGATCAAATGGTT 1
AACGATCGTTCCTGA 1
AACGATCGTTGGATC 2

Total number of rows: 169172

Table truncated, full table size 4287 Kbytes.




Supplementary data files not provided
SRA Run SelectorHelp
Raw data are available in SRA
Processed data included within Sample table

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