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Sample GSM1830092 Query DataSets for GSM1830092
Status Public on Feb 19, 2016
Title pulse_array_bio1
Sample type RNA
Source name Bcell+Cebpa18h
Organism Mus musculus
Characteristics strain: C57/Bl6
tissue: bone marrow
cell type: Bcell
Treatment protocol For the reprogramming experiments B cells were exposed for 18h to 100nM of β-Estradiol (E2) in gelatinized plates seeded with a feeder layer of the MEFs in RPMI medium. After E2 washout, the cultures were switched to serum-free N2B27 medium supplemented with 2μg/ml of doxycycline, IL-4 10ng/ml, IL-7 10ng/ml and IL-15 2ng/ml. B cells were seeded at a density of 500 cells/cm2 in 12 well plates.
Growth protocol ESCs (E14TG2) were cultured on gelatinized plates in N2B27 media (50 %DMEM/F12, 50% Neurobasal medium, N2 (500X), B27 (1000X)) supplemented with small-molecule inhibitors PD (1 μM, PD0325901), CHIR (3 μM, CHIR99021) and LIF. CD19+ pre-B cells were obtained from bone marrow with monoclonal antibody to CD19 (BD Pharmingen) using MACS sorting (Miltenyi Biotech). The purity of the sorted cell fractions was confirmed by FACS using an LSR2 machine (BD). After isolation, B cells were grown in RPMI medium supplemented with 10% FBS and 10ng/ml IL-7 (Peprotech). All medium contain 100 X L-glutamine, 100X penicillin/streptomycin, 100X nonessential amino acids, 1000X β-mercaptoethanol. All reagents are from Life Technologies unless otherwise specified. Lin- c-Kit+ Sca-1- CD16+/CD32+ CD34+ GMP cells were isolated by FACS sorting using a BD INFLUX sorting machine and culture in STEMSPAN medium (Stemcell technologies) supplemented with 100ng/ml SCF, 50ng/ml IL3, 50ng/ml Flt3L and 50ng/ml mTPO (all from Peprotech). The antibodies used from GMPs isolation were from BD Biosciences.
Extracted molecule total RNA
Extraction protocol RNA isolation was performed with the miRNeasy mini kit (Qiagen) following manufacturers instruction. To remove the feeders, cells were pre-plated for 30 minutes before RNA processing. RNA was eluted from the columns using RNase-free water and quantified by Nanodrop. cDNA was produced with the High Capacity RNA-to-cDNA kit (Applied Biosystem).
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 Jul 22, 2015
Last update date Feb 20, 2016
Contact name samuel Collombet
Organization name Ecole Normale Superieure
Department Biology
Lab Thieffry
Street address 46 rue d'Ulm
City Paris
ZIP/Postal code 75005
Country France
Platform ID GPL10787
Series (2)
GSE71213 How C/EBPa creates an elite cell state for reprogramming to pluripotency [array]
GSE71218 How C/EBPa creates an elite cell state for reprogramming to pluripotency

Data table header descriptions
VALUE normalized log2 intensity values

Data table
GE_BrightCorner 14.97489748
DarkCorner 4.149251629
A_55_P2051983 5.331011965
A_52_P169082 5.654619004
A_30_P01028193 4.452534277
A_52_P237997 4.311379443
A_51_P414243 12.07879099
A_55_P2136348 4.839906595
A_51_P108228 3.943045493
A_30_P01033363 6.348053619
A_55_P2049737 4.235936286
A_30_P01024440 8.250191331
A_30_P01025554 14.29089969
A_30_P01031558 5.38704917
A_30_P01030675 4.518715498
A_51_P328014 12.7946325
A_30_P01019108 8.400457339
A_55_P2056220 11.96236233
A_55_P1985764 16.21809321
A_52_P108321 8.884737526

Total number of rows: 55821

Table truncated, full table size 1412 Kbytes.

Supplementary file Size Download File type/resource
GSM1830092_Bcell_serumFree_Cebpa18h_GEM_bio1_141120.txt.gz 12.1 Mb (ftp)(http) TXT
Processed data included within Sample table

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