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Sample GSM787935 Query DataSets for GSM787935
Status Public on Nov 16, 2011
Title BJAB expressing miR-K4-3p_replicate 2
Sample type RNA
 
Channel 1
Source name human B cell line BJAB stably transduced with pNL-SIN-CMV-AcGFP/miR-K4-3p
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) e-gottwein@northwestern.edu
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

Data table header descriptions
ID_REF
VALUE nomalized log2 ratio (sample/reference)

Data table
ID_REF VALUE
H200000005 -0.04632134
H200000010 3.152205
H200000011 -1.2990246
H200000014 2.6687453
H200000016 0.49617666
H200000021 -6.0366945
H200000022 0.20382088
H200000023 -0.5546503
H200000024 2.552826
H200000025 -0.28710976
H200000034 0.39160505
H200000035 -0.052345473
H200000039 -0.22930697
H200000040 1.0614204
H200000042 1.7222762
H200000045 -2.4261992
H200000047 -7.782901
H200000049 -0.506686
H200000051 0.65070766
H200000053 -0.67427504

Total number of rows: 19323

Table truncated, full table size 410 Kbytes.




Supplementary file Size Download File type/resource
GSM787935.gpr.gz 3.3 Mb (ftp)(http) GPR
Processed data included within Sample table
Processed data are available on Series record

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