|
| Status |
Public on Jun 02, 2019 |
| Title |
ChIP-WT-DNMT3B-rep1 |
| Sample type |
SRA |
| |
|
| Source name |
H1 human embryonic stem cell
|
| Organism |
Homo sapiens |
| Characteristics |
tissue: human embryonic stem cell
mutation: Wild type
passage: 44-60
antibody: anti-human DNMT3B rabbit monoclonal antibody (Clone D3C8T, Cell Signaling Technologies, custom-made antibody)
|
| Treatment protocol |
DNMT3B knock out and PWWP domain deletion was achived using CRISPR-Cas9 gene editing method.
|
| Growth protocol |
H1 cell and derivatives were cultured on hESC-qualified Matrigel (354277, Corning) coated plate and were fed with mTeSR1 (05850, Stemcell Technologies) daily. The cell were passage every 4 to 6 days.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
1) ChIP-seq:Cell were fixed with 1% formaldehyde and insoluble chromatin fraction were isolated. Chromatin were sonicated to 100 to 500 base pair and protein-DNA complexes were pulled down by antbodies. 2) RNA-seq: RNA was harvested by trizol followed by DNAseI treatment and Qiagen RNA mini column for further purification. 3) WGBS: gDNA was harvested by lysing the cells in 50mM Tris-HCl pH8, 100mM NaCl, 25mM EDTA and 1%SDS buffer with 1mg/mL of proteinase K and incubate at 65C overnight.
1) ChIP-seq: Libraries were prepared using ThruPLEX DNA-seq kit (R400428) according to manufacturer's instruction. Constructed libraries were PAGE purified and only libraries with 250 to 500bp were used for sequencing 2) RNA-seq: Librarries were prepared using Scriptseq V2 RNA-seq Library preparation kit (SSV21124) according to manufacturer's instruction. Constructed libraries were PAGE purified and only libraries with 250 to 500bp were used for sequencing. 3) WGBS library were prepared by BGI using standard Illumina WGBS protocol.
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2500 |
| |
|
| Data processing |
For ChIP-seq analysis, reads were mapped by bowtie2 against human reference genome GRCh38 (Langmead and Salzberg 2012).
The resulting bam files are converted to bigWig format by bedGraphToBigWig from bedGraph generated by bedtools.
Supplementary_files_format_and_content: bigWig format
For WGBS analysis, the leading 3 bases and adaptor sequences were trimmed from paired-end reads by TrimGalore.
The resulting FASTQ files were analyzed by BISMARK.
PCR duplicates were removed by SAMtools rmdup.
Then bismark_methylation_extractor continued the extraction of the DNA methylation status on every cytosine sites.
Supplementary_files_format_and_content: txt file recoding cytosine methylation status
For RNA-seq analysis, the paired-end RNA-seq reads were trimmed for adaptor sequence by TrimGalore and then mapped by STAR to the human reference genome GRCh38 with reference gene annotation GENCODE 26
PCR duplicates were removed in the paired-end alignments by samtools rmdup.
Alignments with mapping quality < 20 were removed.
Gene expression levels in FPKM were determined by cuffdiff in the Cufflinks package
Genome_build: hg38
Supplementary_files_format_and_content: txt file recoding FPKM values
|
| |
|
| Submission date |
Apr 30, 2018 |
| Last update date |
Jun 03, 2019 |
| Contact name |
Jia Li |
| Organization name |
Duke-NUS
|
| Street address |
8 College Road
|
| City |
Singapore |
| ZIP/Postal code |
169857 |
| Country |
Singapore |
| |
|
| Platform ID |
GPL16791 |
| Series (1) |
| GSE113881 |
DNMT3B maintains mCA landscape and regulates mCG status of bivalent promoters in human embryonic stem cells |
|
| Relations |
| BioSample |
SAMN09005004 |
| SRA |
SRX4015270 |
