|
| Status |
Public on Sep 10, 2015 |
| Title |
NIH3T3_RRBS_EP10 |
| Sample type |
SRA |
| |
|
| Source name |
NIH3T3_RRBS_14-3-3ε-overexressing clone
|
| Organism |
Mus musculus |
| Characteristics |
cell line: NIH3T3
cell type: Mouse embryonic fibroblast cell line
clone id: 14-3-3-epsilon clone 10
genotype/variation: overexressing the 14-3-3-epsilon homologue
|
| Treatment protocol |
14-3-3ε was cloned into p3xFLAG-CMV-10 vector (Sigma-Aldrich). NIH3T3 stable cell line overexpressing 14-3-3ε was generated by selecting the cells transfected with 14-3-3ε constructs in 800 μg/mL of G418. After a week in culture single colonies were picked up and verified by western blotting using anti-FLAG antibody.
|
| Growth protocol |
NIH3T3 stable clones were cultured in DMEM media supplemented with 10% of Fetal Bovine Serum.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Genomic DNA were extracted using Easy-DNA™ gDNA Purification Kit (Life Technologies). Total RNA were extracted using TRIZol reagent.
For RRBS, 1 μg of genomic DNA from stable clones overexpressing 14-3-3ε or control vector was digested by MspI, end-repaired and dA-tailed according to manufacturer’s conditions (New England Biolabs). Methylated NEB Illumina loop adaptor was ligated to digested DNA (E7370S, New England Biolabs). Ligation product was size-selected for 150 to 400 bp fragments on 2% agarose gels and bisulfite converted using the EZ DNA Methylation Kit (Zymo Research). Libraries were enriched by PCR using EpiMark Hot Start Taq DNA Polymerase (New England Biolabs). For RNA-seq, libraries were constructed using the NEBNext® Ultra™ Directional RNA Library Prep Kit (New England Biolabs) per the manufacturer’s instructions. Average insert size of libraries was 200 bp.
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| |
|
| Library strategy |
Bisulfite-Seq |
| Library source |
genomic |
| Library selection |
Reduced Representation |
| Instrument model |
Illumina Genome Analyzer II |
| |
|
| Description |
processed data file: RRBS_CpG_methylation_call.txt
|
| Data processing |
For RRBS, adaptor and low quality sequences (Phred score < 20) were trimmed from RRBS reads using the trim_galore package (http://www.bioinformatics.babraham.ac.uk/projects/trim_galore/) with the parameter of --RRBS --paired. Reads were mapped to the mouse reference genome mm10 using Bismark with Bowtie2. CpG methylation was extracted from uniquely mapped reads using Bismark methylation extractor with the parameter of -p --no_overlap. Differential methylation analysis was carried out using the methylKit package, where logistic regression test was implemented on single CpG sites or genomic region sliding windows (window size 5 kb, step size 5 kb). The p-value of differential methylation test was corrected to q-values using the sliding linear model method. CpG sites or genomic regions with q-values < 0.01 were considered significantly differentially methylated.
For RNA-seq, sequencing reads were mapped to mm10 using TopHat2 (-r 60 --library-type fr-firststrand) with a known transcriptome file supplied (UCSC May 23, 2014). The number of reads mapped to each known gene were counted using htseq-count tools (-t exon -s reverse -i gene_id) and analyzed with DESeq2, where a negative binomial generalized linear model test is implemented on raw counts. The p-value of differential expression was corrected using Benjamini-Hochberg adjustment method and adjusted p-value was obtained. Genes with adj p-value < 0.01 were considered significantly differentially expressed. Sample distance was calculated using Euclidean distance on the rlog-transformed counts. Heatmap were generated using the Z-scored-transformed rlog values of significantly changed genes. Gene ontology analysis was carried out using the AmiGO 2 tools. Multiple comparisons were corrected using Bonferroni correction.
Genome_build: mm10
Supplementary_files_format_and_content: RRBS_CpG_methylation_call.txt: tab-delimited text file including the coordinates of CpG sites (1-based), strand, pvalue, qvalue and meth.diff
Supplementary_files_format_and_content: RNA-seq_log2fdc.txt: gene symbol, baseMean, log2FoldChange, lfcSE, stat, pvalue and padj of each gene
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| |
|
| Submission date |
Sep 09, 2015 |
| Last update date |
May 15, 2019 |
| Contact name |
Guoqiang Zhang |
| E-mail(s) |
zhangg@neb.com
|
| Organization name |
New England Biolabs Inc
|
| Street address |
240 County Road
|
| City |
Ipswich |
| State/province |
Massachusetts |
| ZIP/Postal code |
01938 |
| Country |
USA |
| |
|
| Platform ID |
GPL9250 |
| Series (1) |
| GSE72860 |
Binding of 14-3-3 reader proteins to phosphorylated DNMT1 facilitates aberrant DNA methylation and gene expression |
|
| Relations |
| BioSample |
SAMN04044129 |
| SRA |
SRX1213229 |
