Comment[ArrayExpressAccession] E-GEOD-57419 MAGE-TAB Version 1.1 Public Release Date 2014-06-24 Investigation Title Genome-wide analysis of next generation sequencing for Lsh+/+ and Lsh-/- mouse embryonic fibroblasts Comment[Submitted Name] Genome-wide analysis of next generation sequencing for Lsh+/+ and Lsh-/- mouse embryonic fibroblasts Experiment Description To examine the relationship of reduced CG methylation and gene expression in Lsh KO MEFs, we computed mean CG methylation levels at promoter regions of protein-coding genes. About 60% of TSS regions of protein-coding genes display a difference of CG methylation values greater than 0.3 (WT CG methylation minus KO CG methylation) indicating that Lsh deletion has widespread effects at promoter regions. RNA-seq analysis detects similar transcript steady state levels in WT and KO samples. To determine the relationship of Pol II binding and CG methylation reduction in KO MEFs, Pol II Chip-seq was performed. Protein coding genes were ranked according their CG methylation differences between WT MEFs and KO MEFs. The greatest loss of CG methylation is found at promoter with low CG density. Pol II association is inversely related to the number of CpG sites within promoter regions. KO MEFs show less Pol II association at CG rich promoter regions. However, RNA-seq reads are indistinguishable comparing WT and KO samples, suggesting similar transcriptional efficiency in the absence of Lsh. To explore other molecular mechanisms that may preserve low transcription activity or repression at CG hypomethylated promoter regions, we examined H3K27me3 and H3K4me3 modifications by ChIP-seq. Genome wide computation of histone modifications at 5kb tiles shows no increase of H3K27me3 level in KO MEFs. When we ranked 5kb tiles based on CG methylation differences between WT and KO, we observed alterations in H3K27me3 distribution, while H3K4me3 modifications are unremarkable. Regions with moderate CG methylation reduction exhibit concomitant decreases in H3K27me3. mRNA profiles and Genome-wide maps of H3K27me3, H3K4me3 and Pol II in wildtype (WT) and Lsh KO primary MEFs. 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 Muegge Yu Han Ren Landsman McIntosh Lister Zhu Lee Briones Terashima Leighty Person First Name Kathrin Weishi Yixing Jianke David Carl Ryan Iris Eunice Victorino Minoru Robert Person Email Kathrin.Muegge@nih.gov Person Affiliation National Cancer Institute Person Phone 3018461386 Person Address MCGP, National Cancer Institute, 1050 boyles st, Frederick, MD, USA Person Roles submitter Protocol Name P-GSE57419-4 P-GSE57419-1 P-GSE57419-3 P-GSE57419-2 Protocol Description ChIP-seq, RTA 1.8.70.0 software was used for basecall analysis and aligned to the reference genome was performed at a mismatch error rates =<0.05%. CASAVA1.8.2 (Eland_extended used for mapping) Tag density was performed using IGV tool at a window of 5kb and selected at an FDR<0.0001 for autosomes only. RNA-seq, RTA 1.12.4.2 software was used for basecall analysis and aligned to the reference genome was performed at a mismatch error rates =<0.05%. CASAVA1.8.2 (Both eland_pair and eland_rna module were used for mapping) To evaluate the abundance of transcripts, each set of WT and KO MEF samples and their biologic replica were analyzed independently using Partek Genomics Suite software. Reads per kilobase of exon per million mapped reads (rpkm) were calculated for all murine genes. Reads that mapped to multiple locations in the genome were discarded. Genome_build: mm9 Supplementary_files_format_and_content: Wig files with tag density for ChIP-seq data, txt with RPKM for RNA-seq data. Primary MEF cells were derived from the body of E13.5 C57Bl/6 mouse embryos and genotyping was performed to select Lsh WT and Lsh KO. Total RNA was isolated from cell pellets of WT (Lsh+/+) MEFs and KO (Lsh-/-) MEFs and their biologic replica using the RNeasy Mini Kit (Qiagen) and treated with DNaseI (Roche) for 10 min at room temperature. rRNAs were removed from 5 μg of total RNA by RiboMinus (Life Technologies) as per the manufacturer’s instructions. Cells were cross-linked with formaldehyde and nuclear DNA was sonicated with 200-300bp, then the H3K4me1-DNA complexes were isolated with antibody. The poly-A containing mRNA molecules were purified using poly-T oligo-attached magnetic beads. After purification and PCR amplification the final cDNA library was generated based on the mRNA-seq Library Preparation Protocol from Illumina. ChIP DNA libraries were made following Illumina ChIP-Seq library preparation kit and subjected to Solexa sequencing (Illumina) at the CCR-Sequencing Facility, National Cancer Institute. Sequencing was performed on Illumina Genome Analyzer IIx. Lsh WT and Lsh KO MEFs were maintained in DMEM supplemented with 10% (vol/vol) FBS (Gibco), 2 mM Glutamax (Invitrogen), and 1% penicillin and streptomycin (Invitrogen). Protocol Type normalization data transformation protocol sample treatment protocol nucleic acid library construction protocol growth protocol Experimental Factor Name CHIP ANTIBODY GENOTYPE Experimental Factor Type chip antibody genotype Comment[SecondaryAccession] GSE57419 Comment[GEOReleaseDate] 2014-06-24 Comment[ArrayExpressSubmissionDate] 2014-05-07 Comment[GEOLastUpdateDate] 2014-09-03 Comment[AEExperimentType] RNA-seq of coding RNA Comment[AEExperimentType] ChIP-seq Comment[AdditionalFile:Data1] GSE57419_README.txt Comment[SecondaryAccession] SRP041766 Comment[SequenceDataURI] http://www.ebi.ac.uk/ena/data/view/SRR1274817-SRR1274818 Comment[SequenceDataURI] http://www.ebi.ac.uk/ena/data/view/SRR1274821-SRR1274838 SDRF File E-GEOD-57419.sdrf.txt