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Sample GSM1359925 Query DataSets for GSM1359925
Status Public on Apr 01, 2014
Title B cells, time 24, technical replicate 2
Sample type RNA
 
Source name T24
Organism Mus musculus
Characteristics tissue: primary B cells
time point: 24 hrs
treatment: B cells induced with OSKM for 24hrs
Treatment protocol Cells were trypsinized and FACS-sorted to remove feeder and dead cells.
Growth protocol ESCs and iPSCs were cultured on mitomycin C treated MEF feeder cells in KO-DMEM (Invitrogen) supplemented withL-glutamine, penicillin/streptomycin, nonessential amino acids, β-mercaptoethanol, 1,000 U/ml LIF (ESC medium) and 15% fetal bovine serum (FBS, Invitrogen). MEF cultures were established by trypsin digestion of mouse embryos (embryonic day 13.5) and the resulting cells cultured in DMEM supplemented with 10% FBS, L-glutamine and penicillin/streptomycin. CD19+ pro-B and pre-B cells were isolated from bone marrow using monoclonal antibodies to CD19 (1D3), obtained from BD Pharmingen, using MACS (Miltenyi Biotech). After isolation B cells were grown in RPMI medium supplemented with 10% FBS and IL-7. cell reprogramming experiments were conducted in gelatinized plates seeded with a feeder layer of the OP9 stromal cell line, using ESC medium supplemented with 2μg/ml of doxycycline and 15% FBS. For the reprogramming of B cells, IL-4 (10ng/ml), IL-7 (10ng/ml) and IL-15 (2ng/ml) were added to the medium.
Extracted molecule total RNA
Extraction protocol RNA isolation was done with the miRNeasy Mini kit
Label cy3
Label protocol Cyanine-3 (Cy3) labeled cRNA was prepared from 100ng RNA using the LowInputQuick Amp Labeling kit (Agilent 5190-2305) according to the manufacturer's instructions, followed by RNAeasy column purification (QIAGEN, Valencia, CA). Dye incorporation and cRNA yield were checked with the NanoDrop ND-1000 Spectrophotometer.
 
Hybridization protocol 600ng of Cy3-labelled cRNA (specific activity >10.0 pmol Cy3/ug cRNA) was fragmented at 60°C for 30 minutes in a reaction volume of 25 ul containing 1x Agilent fragmentation buffer and 2x Agilent blocking agent following the manufacturers instructions. On completion of the fragmentation reaction, 25ul of 2x Agilent hybridization buffer was added to the fragmentation mixture and hybridized to Agilent SurePrint G3 Human Gene Expression 8x60K arrays (G4851A-028004) for 17 hours at 65°C in a rotating Agilent hybridization oven. After hybridization, microarrays were washed 1 minute at room temperature with GE Wash Buffer 1 (Agilent) and 1 minute with 37°C GE Wash buffer 2 (Agilent)
Scan protocol Scanned on an Agilent G2539A scanner at 3um resolution and 100%PMT. The intensity data of each individual hybridization were extracted and the quality was assessed with the Feature Extraction software 10.7 (Agilent).
Data processing Raw data was corrected for background noise using the normexp method. Quantile normalization was applied to assure comparability across samples.
 
Submission date Mar 31, 2014
Last update date Apr 01, 2014
Contact name Bruno Di Stefano
E-mail(s) distefanob@gmail.com
Organization name Harvard University
Department Harvard Dept. of Stem Cell and Regenerative Biology
Street address 185 Cambridge Street
City Boston
State/province Ma
ZIP/Postal code 02114
Country USA
 
Platform ID GPL13912
Series (2)
GSE46321 C/EBPα poises B cells for rapid reprogramming into iPS cells [array]
GSE52397 C/EBPα poises B cells for rapid reprogramming into iPS cells

Data table header descriptions
ID_REF
VALUE normalized log2 intensity values

Data table
ID_REF VALUE
1 12.57307462
2 6.05423439
3 6.025783844
4 6.235733939
5 7.495527785
6 6.623578447
7 6.290029757
8 10.94553094
9 6.218305679
10 6.482923761
11 6.765247318
12 6.029722448
13 9.127199238
14 13.88367941
15 7.3695081
16 5.9500035
17 12.87490305
18 7.099185575
19 12.27463562
20 15.37829427

Total number of rows: 62976

Table truncated, full table size 1089 Kbytes.




Supplementary file Size Download File type/resource
GSM1359925_12_IG_1400252800513235_Cy3_T24_1_4.txt.gz 11.9 Mb (ftp)(http) TXT
Processed data included within Sample table

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