|
| Status |
Public on Nov 16, 2011 |
| Title |
BJAB expressing miR-K1_replicate 3 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
human B cell line BJAB stably transduced with pNL-SIN-CMV-AcGFP/miR-K1
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: BJAB
|
| Treatment protocol |
Lentiviral vectors were produced by transfection of 293T cells and used to transduce about 106 BJAB at a cell concentration of about 5x105/ml. The next day, the growth medium was exchanged; 48 h after transduction, cells were collected by centrifugation and resuspended in medium containing 2mM EDTA. AcGFP-expressing cells were sorted (using the 488-nm line of a 20mW laser) and analyzed with a BD FACSAria cell sorter with DiVa software (BD Biosciences). Cell populations of similar mean fluorescence intensities were collected for all samples. Cell pools were expanded and cytoplasmic RNA for microarray analysis was prepared with the RNeasy Mini kit (Qiagen) and harvested on days 12 or 16 after transduction of BJAB cells.
|
| Growth protocol |
BJAB were cultured in RPMI1640 medium supplemented with 10 mg/ml gentamicin and 10% fetal bovine serum (FBS).
|
| Extracted molecule |
cytoplasmic RNA |
| Extraction protocol |
cytoplasmic RNA for microarray analysis was prepared with the RNeasy Mini kit (Qiagen) according to instructions provided with the kit.
|
| Label |
Cy5
|
| Label protocol |
Cytoplasmic RNA (10 mg) from each sample and the reference (Universal Human Reference RNA; Stratagene) were hybridized to oligo(dT) primers at 65 °C and then incubated at 42 °C for 2 h in the presence of reverse transcriptase, Cy5-dUTP or Cy3-dUTP and Cy5-dCTP or Cy3-dCTP, and a deoxynucleotide mix. In all cases, BJAB-derived RNA samples were labelled with Cy5 and reference samples were labelled with Cy3. NaOH was used to destroy residual RNA.
|
| |
|
| Channel 2 |
| Source name |
Universal Human Reference RNA, Stratagene
|
| Organism |
Homo sapiens |
| Characteristics |
reference: Universal Human Reference RNA, Stratagene
|
| Treatment protocol |
Lentiviral vectors were produced by transfection of 293T cells and used to transduce about 106 BJAB at a cell concentration of about 5x105/ml. The next day, the growth medium was exchanged; 48 h after transduction, cells were collected by centrifugation and resuspended in medium containing 2mM EDTA. AcGFP-expressing cells were sorted (using the 488-nm line of a 20mW laser) and analyzed with a BD FACSAria cell sorter with DiVa software (BD Biosciences). Cell populations of similar mean fluorescence intensities were collected for all samples. Cell pools were expanded and cytoplasmic RNA for microarray analysis was prepared with the RNeasy Mini kit (Qiagen) and harvested on days 12 or 16 after transduction of BJAB cells.
|
| Growth protocol |
BJAB were cultured in RPMI1640 medium supplemented with 10 mg/ml gentamicin and 10% fetal bovine serum (FBS).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
cytoplasmic RNA for microarray analysis was prepared with the RNeasy Mini kit (Qiagen) according to instructions provided with the kit.
|
| Label |
Cy3
|
| Label protocol |
Cytoplasmic RNA (10 mg) from each sample and the reference (Universal Human Reference RNA; Stratagene) were hybridized to oligo(dT) primers at 65 °C and then incubated at 42 °C for 2 h in the presence of reverse transcriptase, Cy5-dUTP or Cy3-dUTP and Cy5-dCTP or Cy3-dCTP, and a deoxynucleotide mix. In all cases, BJAB-derived RNA samples were labelled with Cy5 and reference samples were labelled with Cy3. NaOH was used to destroy residual RNA.
|
| |
|
| |
| Hybridization protocol |
Sample and reference cDNAs were pooled, purified with QIAquick Purification Columns (Qiagen), mixed with hybridization buffer (50% formamide, 5xSSC and 0.1% SDS), COT-1 DNA and polydeoxyadenylic acid to limit non-specific binding, and heated to 95 °C for 2 min. This mixture was pipetted onto a microarray slide and hybridized overnight at 42 °C on the MAUI hybridization system (BioMicro Systems). Arrays were printed at the Duke Microarray Facility using the Genomics Solutions OmniGrid 300 Arrayer. The arrays contain the Human Operon v3.0.2 arrays (Oligo Source) that possess 34,602 unique optimized 70-mers.
|
| Scan protocol |
The array was washed at increasing stringencies and scanned on a GenePix 4000B microarray scanner (Axon Instruments).
|
| Data processing |
All arrays were subjected to background subtraction followed by loess normalization within each array and scale normalization across all arrays with the arrayMagic package in R. The KNN impute package in GenePattern was used to impute missing data if a probe had intensity values for at least half the samples. Otherwise the probes were excluded from analysis. Replicate probes were collapsed to one probe corresponding to the median value of all the replicates.
|
| |
|
| Submission date |
Aug 30, 2011 |
| Last update date |
Aug 13, 2020 |
| Contact name |
Eva Gottwein |
| E-mail(s) |
[email protected]
|
| Organization name |
Northwestern University
|
| Department |
Microbiology-Immunology
|
| Street address |
320 E Superior St., Tarry building, Room 6-752
|
| City |
Chicago |
| State/province |
IL |
| ZIP/Postal code |
60611 |
| Country |
USA |
| |
|
| Platform ID |
GPL5770 |
| Series (2) |
| GSE31746 |
BJAB Cell Lines Transduced with lentiviral vector pNL-SIN-CMV-AcGFP expressing KSHV miRNAs miR-K1, miR-K12-11, or miR-K4-3p |
| GSE32113 |
microRNA Targetome Analysis of Latently KSHV-infected Primary Effusion Lymphoma Cell lines Using PAR-CLIP |
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