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| Status |
Public on May 12, 2019 |
| Title |
negative_siRNA_BS_rep1 (2) |
| Sample type |
SRA |
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| Source name |
Embryonic cardiomyocyte
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| Organism |
Mus musculus |
| Characteristics |
strain: CD-1
tissue: cardiomyocyte
developmental stage: embrynoic day 13.5
treatment: negative siRNA
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| Treatment protocol |
Cardiomyocytes were cultured for 48 h at 37°C to reach 70-80% confluency. Cells were transfected with either Qiagen FlexiTube GeneSolution (with 4 siRNAs) DNMT1 siRNA (#GS13433) or AllStars negative control siRNA (#1027281) at 12 nM using lipofectamine RNAiMAX (n=3 per treatment).
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| Growth protocol |
Primary cardiomyocytes were isolated from mouse E13.5 embryonic ventricles. Briefly, hearts were dissected from embryos, and the ventricular tissue from each litter of embryos was pooled and digested with papain solution that contained 4 mg/ml papain, 1.1 mM EDTA, 5.5 mM L-cystein, and 67 mM b-mercaptoethanol dissolved in the Earle’s Balanced Salt Solution. Digestion was terminated with 1 mg/ml soybean trypsin inhibitor solution. Cells were suspended in DMEM supplemented with 10% inactivated fetal bovine serum, 2 mM L-glutamine, and antibiotic/antimycotic solution. Cells were placed in non-coated flasks for 1 h to allow differential attachment to occur. The non-attached cells, which are predominantly cardiomyocytes, were transferred to new flasks coated with 2% gelatin.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
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), followed by magnetic sorting 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. Bisulfite specific PCR primers for fifteen target genes (two promoter regions per gene) were designed with Methyl Primer Express V1.0 (Thermo Fisher Scientific, Waltham, MA). Target gene regions were PCR-amplified from the bisulfite-converted DNA with ZymoTaq™ PreMix (Hot start DNA taq polymerase, ZYMO Research). 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.
PCR products from the same genomic DNA were made into an indexed sequencing library with the Nextera XT DNA Library Preparation kit and the Nextera XT Index Kit (Illumina). Briefly, amplicon pools were tagmented, PCR-amplified with index primers, and cleaned with AMPure XP beads. The libraries were validated with an Agilent 2200 TapeStation high sensitivity DNA assay and quantitated by a KAPA Library Quantification kit (Illumina platforms; Kapa Biosystems) on a GeneAmp 7300 Real Time PCR (Thermo Fisher Scientific).
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| Library strategy |
Bisulfite-Seq |
| Library source |
genomic |
| Library selection |
RANDOM |
| Instrument model |
Illumina MiSeq |
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| Data processing |
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.
Genome_build: mm10
Supplementary_files_format_and_content: Tab-delimited text files include percent methylation for each cytosine in the target gene promoters.
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| Submission date |
May 16, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
Xiefan Fang |
| E-mail(s) |
xiefanfang@ufl.edu
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| Organization name |
University of Florida
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| Department |
Pediatrics
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| Street address |
1200 Newell Drive, ARB R1-148
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| City |
Gainesville |
| State/province |
Florida |
| ZIP/Postal code |
32610 |
| Country |
USA |
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| Platform ID |
GPL16417 |
| Series (1) |
| GSE81464 |
Multiplex bisulfite sequencing identifies differentially methylated regions in target genes in embryonic cardiomyocytes following knockdown of DNMT1 expression |
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| Relations |
| BioSample |
SAMN05001435 |
| SRA |
SRX1763604 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2152810_negative_siRNA_BS_rep1_count.txt.gz |
55.4 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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