Computational protocol: Tet-mediated imprinting erasure in H19 locus following reprogramming of spermatogonial stem cells to induced pluripotent stem cells

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Protocol publication

[…] In order to minimize the contamination with the feeder cells, iPSC at passages 13-14 were purified by MACS for SSEA1 following a protocol similar to the one described for SSC enrichment for Gfra1, but using anti-SSEA-1 (CD15) microbeads (Miltenyi Biotech). The purified cells were used for gene expression and DNA methylation analysis. qPCR was performed as previously described and differences between groups were analyzed by ANOVA (SigmaStat). Global transcriptional differences between SSC, SSCiPSC and fiPSC were analyzed by deep sequencing of RNA libraries. Total RNA was extracted with miRNeasy Minikit following manufacturer instructions. RNA concentration, purity and integrity were verified by Nanodrop (Eppendorf) and Nanochip on Bioanalyzer (Agilent). The conversion of the mRNA in total RNA into a library of template molecules suitable for subsequent cluster generation and DNA sequencing was performed following the TruSeq® Stranded mRNA kit (Illumina, San Diego, CA, USA). Libraries were quantified by Quant-iT PicoGreen followed by qPCR. Libraries are clustered using TruSeq PE Cluster Kit v3 – cBot HS, and sequenced using TruSeq SBS Kit v3 - HS (200-cycles). Paired end 100 bp reads were processed and analyzed using Cufflinks and Tophat software platforms. Statistical analysis was performed using Cummerbund R software. Gene Ontology analysis was performed using DAVID Functional Annotation Tools (http://david.abcc.ncifcrf.gov/), and differentially expressed genes with p-value ≤ 5*10−5 and fold change ≥ 1.5 were categorized with respect to Molecular Function, Biological Process and Cellular Component. The annotated genes were also mapped into relevant functional groups in a pathway analysis according to the Kyoto Encyclopedia of Genes and Genomes (KEGG). […]

Pipeline specifications

Software tools Cufflinks, TopHat, CummeRbund, DAVID
Databases KEGG
Application Transcriptome data visualization
Organisms Mus musculus
Diseases Teratoma