Comment[ArrayExpressAccession] E-GEOD-41912 MAGE-TAB Version 1.1 Public Release Date 2012-11-20 Investigation Title Transcriptome profiling of wild type and Tet1-null primordial germ cells (PGC) by Bisulfite-seq Comment[Submitted Name] Transcriptome profiling of wild type and Tet1-null primordial germ cells (PGC) by Bisulfite-seq Experiment Description Purpose: To investigate the effect of Tet1 depletion on global DNA methylation, we performed whole-genome bisulfite sequencing (WGBS). Methods: Starting with as little as 1400-5251 manually micro-dissected PGCs, we used an ultra-low input method, Tn5mC-seq. Results: We generated 945 million reads for Tet1Gt/Gt PGCs and 302 million reads for wild-type PGCs. We obtained 14-16 million CpG sites per genotype at 1.76-2.66x genome coverage, which enables a comprehensive view of genome-wide DNA methylation patterns in E13.5 PGCs. PGCs are almost completely unmethylated genome-wide. Loss of Tet1 led to a subtle increase of methylation level in various genomic elements including promoters, exons, introns and repetitive elements in Tet1Gt/Gt PGCs (p<0.01). Local analysis identified 4,337 differentially methylated regions (DMRs) between Tet1-/- PGCs and wild-type cells. These DMRs are associated with 5,261 genes, among which 271 genes also exhibited differential gene expression and enriched for the cell cycle pathway (FDR=0.02). Conclusions: This result revealed that demethylation of certain set of cell cycle genes is largely abolished in the Tet1-/- PGCs. Genome-wide methylation profiles of primordial germ cells derived from the wild type (WT) and Tet1-null female embryos at E13.5 were generated by whole genome bisulfite sequencing using Illumina Hiseq. Term Source Name ArrayExpress EFO Term Source File http://www.ebi.ac.uk/arrayexpress/ http://www.ebi.ac.uk/efo/efo.owl Person Last Name Diep Person First Name Dinh Person Mid Initials Hue Person Email hdinhdp@gmail.com Person Affiliation University of California, San Diego Person Address Bioengineering, University of California, San Diego, 9500 Gilman Dr Mailcode: 0412, La Jolla, CA, USA Person Roles submitter Protocol Name P-GSE41912-1 P-GSE41912-2 Protocol Description gDNA was isolated by Wizard kit (Promega) when > 5,000 cells were available, whereas samples with <5,000 cells were directly lysed in a hypotonic lysis buffer (10mM Tris-HCl pH 7.5; 10mM NaCl; 3mM MgCl2) with 1% NP40 and 0.2 Au/ml protease (Qiagen). Resultant DNA were fragmented and attached with a 5’ methylated adaptor by Tn5 transposon based tagmentation. A 3’ methylated adaptor was added during gap-filling and ligation. Tagged DNA fragments were bisulfite converted by Imprint kit (Sigma) or Lightning kit (Zymo), followed by PCR amplification with Illumina adaptors. Barcoded libraries were pooled and sequenced with Illumina Hiseq 2000 instrument. Reads were aligned to the mouse reference genome (mm9) using SOAP2. Libraries processed with the Sigma Imprint kit contain a mixture of fully converted reads and poorly converted reads. A post-mapping filtering was performed on libraries produced using Sigma Imprint kit to remove unconverted reads based on the criterion that cytosine in the non-CpG context is present at <1.2% in the raw sequencing reads, which ensured a bisulfite conversion rate of >98% in the filtered reads, as estimated by the percentage of un-methylated cytosines on the mitochondrial chromosome (chrM). Libraries processed with the Zymo lightning kit were fully converted, such that the post-mapping filtering was not necessary. Data from the two batches of libraries were pooled since global analysis on the two batches yielded consistent results. The global methylation distribution plot was generated using the average methylation levels of CpGs in 10kb non-overlapping bins along the genome. DMRs (differentially methylated regions) between genotypes were identified by comparing the fraction of methylated cytosines in a sliding window that contains at least six CpG sites based on chi-square test. Repetitive regions were masked prior to DMR calling. Adjacent candidate DMRs (<250bp apart) were joined, and the flanking non-informative CpG sites were further trimmed. We required that there are at least 20 methylated or unmethylated cytosine observations for each sample in a DMR, and the mean methylation difference is at least 0.2. False discovery rate (FDR) was empirically estimated to be ~10%, by applying the same procedure on randomly permutated data sets in which the methylation level of each CpG site was randomly assigned to a different CpG site in the genome while maintaining the global distribution of CpG methylation in each sample. Identified DMRs were correlated with Tet1 binding sites defined in ESCs 17 by requiring >1bp overlapping using BEDtools (http://code.google.com/p/bedtools/). FDR (false discovery rate) was then tested by 100 permutations of randomly placed DMRs along the genomes. DMRs were assigned to potential target genes and functional characterized by GREAT (http://great.stanford.edu/public/html/splash.php). For visualization, locally smoothed methylation levels were calculated by the Bsseq package with parameter of ns = 40, h = 2000 to obtain high confidence methylation levels for each genotypes Genome_build: mm9 Supplementary_files_format_and_content: BED formated (tab-delimited) text files include chromosome position and methylation fraction value for each CpG in each sample. Protocol Type nucleic acid library construction protocol feature_extraction Experimental Factor Name BISULFITE CONVERSION PROTOCOL INPUT CELL NUMBER GENOTYPE SEQUENCING INFO Experimental Factor Type bisulfite conversion protocol input cell number genotype sequencing info Publication Title Tet1 controls meiosis by regulating meiotic gene expression. Publication Author List Yamaguchi S, Hong K, Liu R, Shen L, Inoue A, Diep D, Zhang K, Zhang Y PubMed ID 23151479 Publication DOI 10.1038/nature11709 Comment[SecondaryAccession] GSE41912 Comment[GEOReleaseDate] 2012-11-20 Comment[ArrayExpressSubmissionDate] 2012-10-29 Comment[GEOLastUpdateDate] 2013-01-30 Comment[AEExperimentType] methylation profiling by high throughput sequencing Comment[AdditionalFile:Data1] GSE41912_PGC-KO-all-chrs.BED Comment[AdditionalFile:Data2] GSE41912_PGC-WT-all-chrs.BED Comment[AdditionalFile:Data3] GSE41912_filter_unconverted_reads.txt Comment[SecondaryAccession] SRP016883 Comment[SequenceDataURI] http://www.ebi.ac.uk/ena/data/view/SRR619106-SRR619136 SDRF File E-GEOD-41912.sdrf.txt