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Sample GSM1572082 Query DataSets for GSM1572082
Status Public on Sep 30, 2016
Title i6F_1
Sample type RNA
 
Source name Foreskin fibroblasts (46,XY), (CRL-2429, ATTC) transduced with the i6F cocktail of genes PRDM1, PRDM14, LIN28A, DAZL, DDX4 and SYCP3 cloned into the lentiviral backbone pLenti7.3-V5
Organism Homo sapiens
Characteristics primary cell culture: Passages 3 to 8
treatment: i6F
cell type: Foreskin fibroblasts
Treatment protocol The Open Reading Frame (ORF) sequences of transgenes were isolated from reverse transcribed adult human testis total RNA (Clontech). Isolated PCR products were cloned into the lentiviral backbone pLenti7.3-V5 (Life Technologies) using the Gateway system (Life Technologies). Lentiviral packaging was generated by transient co-transfection using Lipofectamine 2000 (Life Technologies) of the 293-T packing cell line (CRL-11268, ATCC). The collected viral supernatants supplemented with 8µg/mL polybrene (Sigma Aldrich) were used in a dilution 1:2 with conventional medium for transduction. Remaining viral supernatants were washed off 24h later and GC-M was added the following day.
Growth protocol Briefly, conventional growth medium, DMEM/F12 supplemented with 10% FBS (Life Technologies) and 2mM L-glutamine (Millipore) was used to grow human foreskin fibroblasts (hFSK (46,XY), CRL-2429, ATTC). Induced Germ-like Cells (iGC) were obtained, cultured and maintained in germ cell medium (GC-M), consistent on DMEM/F12 supplemented with 20% KnockOut Serum Replacement (all from Life Technologies), 2 mM L-glutamine, 0.1 mM nonessential amino acids, 0.1 mM 2-mercaptoethanol (Millipore), and supplemented with 1000U/ml human recombinant Leukemia Inhibitor Factor (LIF, Sigma Aldrich), 20ng/mL recombinant human Glial-Derived Neurotrophic Factor (GDNF, Peprotech), 5µM Forskolin (Sigma Aldrich), 10 ng/ml basic Fibroblast Growth Factor (bFGF, Life Technologies), 5ng/mL Stem Cell Factor (SCF, Peprotech) and 2µM retinoic acid (Sigma Aldrich) at 37°C, 5% CO2. All the experiments were performed at least in triplicate using low passages (up to passage 8).
Extracted molecule total RNA
Extraction protocol Total RNA was extracted with the RNAeasy mini kit (Qiagen) according to manufacturer´s instructions. RNA was quantified using a NanoDrop-1000 spectrophotometer and quality was monitored with the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA).
Label Cy3
Label protocol Cyanine-3 (Cy3) labeled cRNA was prepared from 0.2 ug RNA using the One-Color Low RNA Input Linear Amplification PLUS kit (Agilent) 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 1.65 ug of Cy3-labelled cRNA (specific activity >10.0 pmol Cy3/ug cRNA) was fragmented at 60°C for 30 minutes in a reaction containing 1x Agilent fragmentation buffer and 2x Agilent blocking agent following the manufacturers instructions. On completion of the fragmentation reaction, 250 ml of 2x Agilent hybridization buffer was added to the fragmentation mixture and hybridized to Agilent Whole Human Genome Oligo Microarrays (G4845A) 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), then dried immediately by brief centrifugation.
Scan protocol Hybridized microarrays were scanned in an Axon 4100A scanner (Molecular Devices, Sunnyvale, CA), and the data were extracted with the GenePix Pro 6.0 software (Molecular Devices) (Scan Area 61x21.6 mm, Scan resolution 10um, Dye channel is set to Green and Green PMT is set to 100%).
Description Sample name: 251485079649_2-4
Gene expression 14 days post-transduction
Replicate 1
Data processing Gene expression scanned values were preprocessed, normalized, and statistically analyzed. The half-background median intensity values were subtracted from the average intensity of each spot, and were normalized between arrays using the quantile method implemented in the Bioconductor (www.bioconductor.org) LIMMA package run under the R software (www.r-project.org). Probe sets belonging to the same gene were merged by median and transformed to the logarithmic scale (log2) giving rise to a final matrix of 25,512 genes by sample. The microarray analysis was performed using Babelomics software version 4.3 (babelomics.bioinfo.cipf.es) [11]. Exploratory analysis of whole gene expression data was performed to check the behavior of samples using Principal Component Analysis. Functional enrichment for differential expressed genes was performed with FatiGO [12] a widely used SEA implementation, which is included in the Babelomics [11] web-based package using the Gene Ontology (GO) [13] and Reactome curated databases [14]. The significant over-represented terms related to human genome comparison (adjusted p-value< 0.05) were considered. Results were validated by qPCR for three of the top up-regulated and down-regulated genes (data not shown). we applied the LIMMA test for deep screening analysis between groups (two-class comparisons), and compared the expression patterns from treated samples with i12F/i6F versus MOCK controls, obtaining adjusted p-values and fold changes for each comparison (FC higher than 2; adjusted p-value of less than 0.05); we corrected the p values using the false discovery rate accounting for multiple testing effects. Gene expression data fold change from significant genes was used to compare with RT-qPCR values (data not shown).
 
Submission date Dec 23, 2014
Last update date Sep 30, 2016
Contact name Jose Vicente Medrano
E-mail(s) jomepla@gmail.com
Phone +34 677292622
Organization name IIS La Fe
Department Reproduction Unit
Street address Avenida Fernando Abril Martorell, 106
City Valencia
State/province Valencia
ZIP/Postal code 46026
Country Spain
 
Platform ID GPL16022
Series (1)
GSE64479 Human somatic cells subjected to genetic reprogramming with germ line-related factors show meiotic germ cell-like features

Data table header descriptions
ID_REF
VALUE Normalized signal intensity

Data table
ID_REF VALUE
ZBTB10 8.222578
THC2669500 7.599856
TSG101 12.388632
SPG3A 9.375049
SFXN3 10.632484
SNX15 8.114634
MTHFR 8.109965
C11orf2 11.832243
ADORA3 7.844294
A_24_P928815 7.886025
CTAGEP 8.590318
ETS1 8.378414
FLJ38377 8.371302
TERF2 8.301613
AF090926 7.958869
CACNA2D3 9.555414
KIAA1909 7.775857
GRM4 7.90184
GHRL 7.728582
FBXW2 9.399905

Total number of rows: 30499

Table truncated, full table size 520 Kbytes.




Supplementary file Size Download File type/resource
GSM1572082_251485079649_2-4.gpr.gz 4.2 Mb (ftp)(http) GPR
Raw data provided as supplementary file
Processed data included within Sample table

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