GEO Logo
   NCBI > GEO > Accession DisplayHelp Not logged in | LoginHelp
GEO help: Mouse over screen elements for information.
Series GSE77621 Query DataSets for GSE77621
Status Public on Sep 08, 2017
Title Multiplex bisulfite sequencing identifies differentially methylated regions in target genes in embryonic cardiomyocytes following knockdown of DNMT3a or DNMT3b expression
Organism Mus musculus
Experiment type Methylation profiling by high throughput sequencing
Summary Purpose: This study aims to identify the differentially methylated regions in target genes in embryonic cardiomyocytes following the knockdown of DNMT3a or DNMT3b expression. Methods: Primary cardiomyocytes were isolated from mouse embryonic day (E) 13.5 ventricles, and cultured for 48 h at 37°C to reach 70-80% confluency. Cells were transfected with either Qiagen FlexiTube GeneSolution (with 4 siRNAs) DNMT3a siRNA (#GS13435), DNMT3b siRNAs (#GS13436), or AllStars negative control siRNA (#1027281) at 0.012 pmol using lipofectamine RNAiMAX (n=3 per treatment). At 72 h after transfection, cells were released with Accutase (Innovative Cell Technologies, San Diego, CA) and dissociated with Accumax (Innovative Cell Technologies). To sort cardiomyocytes, dissociated cells were stained with anti-VCAM1 antibody conjugated with allophycocyanin (APC; BioLegend, San Diego, CA) and magnetically sorted using anti-APC microbeads and magnetic assisted cell sorting (MACS) columns (Miltenyi Biotec, Bergisch Gladbach, Germany). DNA was extracted with the Quick-gDNA™ MicroPrep kit (Zymo Research, Irvine, CA). Genomic DNA was treated with sodium metabisulfite for bisulfite conversion. Target gene regions for fifteen target genes (two promoter regions per gene) were PCR-amplified from the bisulfite-converted DNA with ZymoTaq™ PreMix (Hot start DNA taq polymerase, ZYMO Research, Irvine, CA). The fifteen genes include Myh6, Myh7, Myh7b, Nppa, Nppb, Tnnc1, Tnni3, Tnnt2, Camta1, Camta2, Cdkn1A, Cdkn1B, Cdkn1C, Mef2c, and Mef2d. PCR products were cleaned with the ZR-96 DNA Clean & Concentrator™-5 kit (ZYMO Research). PCR products from the same genomic DNA sample were pooled at equal concentration ratio and made into an indexed sequencing library with the Nextera XT DNA Library Preparation kit and the Nextera XT Index Kit (Illumina, San Diego, CA). The nine Illumina-adapted libraries were pooled at equal molar ratio, spiked with 20% PhiX control libraries (Illumina), and sequenced with one 1x150 cycles run (v3) on a MiSeq sequencer (Illumina). The fastq files generated from MiSeq were uploaded to the UF Research Computing Galaxy instance developed by the University of Florida. The data were cleaned with the following steps: removed sequencing artifacts, trimmed 5’ or 3’ ends with low scores, removed adaptor contamination, and filtered low quality reads by the FastQC program. A reference genome with the amplicon sequences was built and bisulfite converted in silico with Bismark Bisulfite Mapper. The high quality sequence reads were aligned to the reference genome using Bowtie 2. Cytosine methylation counting was performed with the Bismark methylation extractor. Differential methylation was analyzed with the Methylkit package. CpG sites with false discovery rate (FDR) less than 0.05 and absolute percent methylation difference greater than 5% were considered as significant. Results: Following knockdown of DNMT3a for 72 h, differentially methylated regions were identified in the promoter CpG sites of Myh7, Myh7b, Tnni3, Tnnt2, Nppa, Nppb, Mef2c, Mef2d, and Cdkn1C. Following knockdown of DNMT3b expression for 72 h, differentially methylated regions were identified in the promoter CpG sites of Myh6, Myh7b, Tnni3, Tnnt2, Nppa, Nppb, Mef2d, Cdkn1A, and Cdkn1C.
Overall design The DNA methylation patterns of target promoters in embryonic cardiomyocytes with knockdown of DNMT3a or DNMT3b expression were generated by deep sequencing (n=3 for negative siRNA, n=3 for DNMT3a siRNA, n=3 for DNMT3b siRNA), using Illumina MiSeq.
Contributor(s) Fang X, Rivkees SA, Wendler CC
Citation(s) 27306334
Submission date Feb 05, 2016
Last update date May 15, 2019
Contact name Xiefan Fang
Organization name University of Florida
Department Pediatrics
Street address 1200 Newell Drive, ARB R1-148
City Gainesville
State/province Florida
ZIP/Postal code 32610
Country USA
Platforms (1)
GPL16417 Illumina MiSeq (Mus musculus)
Samples (9)
GSM2055326 negative_siRNA_BS_rep1
GSM2055327 negative_siRNA_BS_rep2
GSM2055328 negative_siRNA_BS_rep3
BioProject PRJNA311045
SRA SRP069340

Download family Format
SOFT formatted family file(s) SOFTHelp
MINiML formatted family file(s) MINiMLHelp
Series Matrix File(s) TXTHelp

Supplementary file Size Download File type/resource
GSE77621_RAW.tar 510.0 Kb (http)(custom) TAR (of TXT)
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

| NLM | NIH | GEO Help | Disclaimer | Accessibility |
NCBI Home NCBI Search NCBI SiteMap