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Sample GSM2026309 Query DataSets for GSM2026309
Status Public on Mar 01, 2016
Title preB_iPS_p20_1 Expression
Sample type RNA
 
Source name preB iPS p20
Organism Mus musculus
Characteristics cell type: preB iPS p20
Treatment protocol Reprogramming experiments with B cells were performed as previously described (Di Stefano et al., 2014); with MEFs, macrophages and NSCs were conducted by plating 100.000 cells/well on gelatinized plates seeded with irradiated MEFs, using ESC medium supplemented with 2 µg/ml of doxycycline. For the isolation of iPSC lines, doxycycline was washed out after 15 days of reprogramming and colonies with ESC-like morphology were picked at 20 days before further passaging. iPSC lines were expanded for an additional 9 days (3 passages) to obtain P3 iPS cell lines or for 20 passages to obtain P20 iPS cell lines.
Growth protocol ESCs and iPSCs were cultured on Mitomycin-C treated MEFs in KO-DMEM (Invitrogen) supplemented with 1% nonessential amino acids (Invitrogen), 0.1 mM β-mercaptoethanol (Invitrogen), 1,000 U/ml LIF (Millipore) and 15% fetal bovine serum (FBS, Invitrogen) (ESC medium). Naïve ESCs were cultured in serum-free N2B27 medium on gelatin-coated dishes. N2B27 medium (500ml) was generated by inclusion of the following: 240ml DMEM/F12 (Invitrogen), 240ml Neurobasal (Invitrogen), 5ml N2 supplement (Invitrogen), 10ml B27 supplement (Invitrogen), 1,000 U/ml LIF (Millipore), 1% nonessential amino acids (Invitrogen), 0.1mM β-mercaptoethanol (Invitrogen), and the small molecules PD0325901 (Stemgent, 1μM) and CHIR (Stemgent, 3μM). CD19+ pre-B cells and Mac1+ macrophages were isolated from bone marrow with monoclonal antibodies to CD19 and Mac-1 (BD Pharmingen) respectively, using MACS sorting (Miltenyi Biotech). B cells were grown in RPMI medium with 10% FBS and 10ng/ml IL-7 (Peprotech); macrophages in DMEM with 10% FBS and 10ng/ml each of CSF1 and IL-3 (Peprotech). MEFs were established from day 13.5 mouse embryos and cultured in DMEM containing 10% FBS. NSC were isolated and cultured as previously described (Di Stefano et al., 2009). All media were supplemented with L-glutamine and penicillin/streptomycin (GIBCO).
Extracted molecule total RNA
Extraction protocol RNA was isolated with the miRNeasy mini kit (Qiagen), eluted with RNase-free water and quantified by Nanodrop. cDNA was produced with the High Capacity RNA-to-cDNA kit (Applied Biosystem). RNA samples with a RIN greater than 9 were analyzed by expression arrays.
Label Cy3
Label protocol 500ng of total RNA per sample were labeled using Agilent's QuickAmp labeling kit and hybridized to Agilent 8X60K expression arrays.
 
Hybridization protocol not provided
Scan protocol not provided
Data processing Raw array data was processed using limma (Ritchie et al., 2015). We perform “normexp” background correction with an offset of 16. We normalize between arrays using quantile normalization. To identify differently expressed genes we create a contrast matrix in which all pairwise comparisons are made. For each probe an empirical Bayes moderated t-statistic test is performed. An FDR correction is applied to the nominal p-values.
 
Submission date Jan 04, 2016
Last update date Mar 01, 2016
Contact name Elzo de Wit
Phone +31 30 2121 800
Organization name Hubrecht Institute
Street address Uppsalalaan 8
City Utrecht
ZIP/Postal code 3584 CT
Country Netherlands
 
Platform ID GPL10787
Series (2)
GSE76480 Cell-of-origin specific 3D genome structure acquired during somatic cell reprogramming [Expression]
GSE76481 Cell-of-origin specific 3D genome structure acquired during somatic cell reprogramming

Data table header descriptions
ID_REF
VALUE Quantile normalized signal intensity

Data table
ID_REF VALUE
GE_BrightCorner 14.36224922
DarkCorner 5.997803292
A_55_P2051983 5.983258543
A_52_P169082 7.28048189
A_30_P01028193 6.579874253
A_52_P237997 6.015560009
A_51_P414243 11.59403188
A_55_P2136348 5.894183394
A_51_P108228 6.995422202
A_30_P01033363 7.140610008
A_55_P2049737 6.077870779
A_30_P01024440 9.938544244
A_30_P01025554 11.94766771
A_30_P01031558 6.705075686
A_30_P01030675 5.859252629
A_51_P328014 13.1107919
A_30_P01019108 8.293328434
A_55_P2056220 11.16204485
A_55_P1985764 16.84314686
A_52_P108321 8.391522905

Total number of rows: 55821

Table truncated, full table size 1413 Kbytes.




Supplementary file Size Download File type/resource
GSM2026309_preB_iPS_p20_1_1400252800519287.txt.gz 12.0 Mb (ftp)(http) TXT
Processed data provided as supplementary file

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