|
| Status |
Public on Apr 01, 2014 |
| Title |
18hrs pulse B cells, time 12, technical replicate 1 |
| Sample type |
RNA |
| |
|
| Source name |
aT12
|
| Organism |
Mus musculus |
| Characteristics |
tissue: primary B cells
time point: 12 hrs
treatment: B cells pulsed for 18hrs with C/EBPa, induced for 12 hrs with OSKM
|
| 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.
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| |
|
| 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.
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| |
|
| 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 |
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