|
| Status |
Public on Jun 16, 2024 |
| Title |
5hmC of Tet1/2/3 triple KO mESC transfected with human Tet2 CD domain rep2 |
| Sample type |
SRA |
| |
|
| Source name |
mESC
|
| Organism |
Mus musculus |
| Characteristics |
cell type: stem cell
|
| Treatment protocol |
Tet1/2/3 triple knock-out mESCs were generated by the CRISPR/Cas9 technology, FLAG-tagged Tets catalytic domain (TET-CD) or deficient with LCI are transfected to 293T with lipofectimin or transduced to mESC through lentivirus.
|
| Growth protocol |
mESCs were cultured on MEFs in Knock-out Dulbecco’s Modified Eagle’s Medium (Gibco), supplemented with 15% fetal bovine serum (Omega), 0.5% penicillin-streptomycin (Gibco), 0.1 mM non-essential amino acids (Gibco), 0.1 mM 2-mercaptoetanol (Sigma), and 103 U/mL of leukemia inhibitory factor (Millipore)
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
DNA were purified using Qiagen AllPrep DNA/RNA micro kit.
CMSIP: 5hmC-containing genomic DNA were immunoprecipitated by CMS antibody after being bisulfite treated. Immunoprecipitated DNA and input were proceed for Library generation by using zymo pico methyl kit.
CMSIP
|
| |
|
| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina NextSeq 500 |
| |
|
| Data processing |
For CMS-IP-seq analysis, raw FASTQ files were mapped to mm10 using BSMAP . Only uniquely mapped non duplicated reads were kept as input for MACS2 to call 5hmC enriched regions (peaks). Differential hydroxymethylation regions (DHMRs) were identified using DESeq2 with default parameters. Bam files were transformed to bigwig format, which can be used to visualize 5hmC signals in the UCSC genome browser.. H3K9me3 and H3K9me2 ChIPseq data was aligned on mm10 using bowtie2. The bam files were inputted to EDD (https://github.com/CollasLab/edd) to identify ChIPseq enriched regions.
For the WGBS analysis, raw FASTQ files were mapped to the NCBI Human Reference Genome Build GRCh37 (hg19) using BSMAP. Fastp was used to trim adaptor sequences from fastq data and remove 15 bp from the two ends of the reads based on Swift protocol. MCALL module in MOABS software was used to calculate DNA methylation ratio and coverage for each CpG site. Differentially methylated CpGs and regions were identified using the MCOMP module considering variance among samples (--withVariacne 1) in the MOABS software. Bisulfite conversion efficiencies were estimated using spike-in unmethylated lambda phage DNA. The output bedGraph files from MCALL include single base resolution DNA methylation ratios, which were transformed to a bigwig file format. The bigwig files were uploaded to the UCSC genome browser for visualization.
Genome_build: mm10
Supplementary_files_format_and_content: bigwig
|
| |
|
| Submission date |
Sep 10, 2021 |
| Last update date |
Jun 16, 2024 |
| Contact name |
Jia Li |
| E-mail(s) |
jiali@tamu.edu
|
| Organization name |
Texas A&M U Health Science Center
|
| Department |
CEDP
|
| Lab |
Jia Li Lab
|
| Street address |
2121 W HOLCOMBE BLVD
|
| City |
Houston |
| State/province |
Texas |
| ZIP/Postal code |
77030 |
| Country |
USA |
| |
|
| Platform ID |
GPL19057 |
| Series (1) |
| GSE183934 |
Perturbation of TET2 condensation induces genome-wide promiscuous DNA hypomethylation and curtails leukemia cell growth |
|
| Relations |
| BioSample |
SAMN21387033 |
| SRA |
SRX12136128 |
