 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on Apr 23, 2023 |
| Title |
WGBS-E7.5-Dnmt1-KO-3 |
| Sample type |
SRA |
| |
|
| Source name |
embryo
|
| Organism |
Mus musculus |
| Characteristics |
developmental stage: E7.5
genotype: Dnmt1-KO
strain: C57BL/6(male) X F1(female)
tissue: whole embryo
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
For RNA-seq, the embryos (E6.5 and E7.5) of control, Dnmt1 knock-out, Dnmt3a/3b double knock-out and Dnmt1/3a/3b triple knock-out were carefully collected from mouse decidua. The epiblast (epi) and extraembryonic ectoderm (exe) of embryos were quickly separated by needle. For WGBS, gDNA was extracted from the embryos (E6.5 and E7.5) by using TIANamp Micro DNA Kit (TIANGEN, Cat#DP316) according to the manufacturer's instructions.
For RNA-seq, the cDNA library experimental procedure was mainly according to reported protocol (Peng et al., 2019, Nature). Sequencing was performed on the Illumina Nova Seq 6000 at the Novogene Bioinformatics Institute, Beijing, China. For WGBS, the DNA methylation library experimental procedure was mainly modified from previous published method (PMID 28182018; PMID 22649061). After extraction of mouse embryo genomic DNA, 10 ng of DNA was taken as the start material for DNA methylation library preparation. 0.1 ng lambda DNA for each sample was spiked into the sample to quantify the C-to-T conversion efficiency. The bisulfite treatment was performed using EZ-96 DNA Methylation-Direct MagPrep (Zymo cat#D5044) based on user instruction, and the sample was eluted in 19.5 μL H2O. To synthesize the first stand after bisulfite conversion, 2.5 μL 10× Blue buffer (TIANGEN), 2 μL 10 mM dNTP (TAKARA) and 1 μL 100 mM bio-P5-N6-Oligo 1 (/5Biosg/CTACACGACGCTCTTCCGATCTNNNNNN) were added to the sample and sample was incubated at 65°C for 3 min. Then 1μL Klenow exo– (TIANGEN) was added and the sample was incubated at 4°C for 5 min, +1°C/15 sec to 37°C, 37°C for 30 min. The random priming was repeated 4 times and the free-primer was removed by treating with Exo I (NEB). The biotin-labelled fragments were enriched using Dynabeads M280 Streptavidin beads (Invitrogen) with rotation at room temperature for 45 min. The second strand was synthesized using P7-N9-oligo2 (AGACGTGTGCTCTTCCGATCTNNNNNN) and Klenow exo– (TIANGEN). The final PCR was performed using NEB pre-indexed primer, universal primer, and KAPA HiFi HotStart ReadyMix (KAPA) for 5 cycles. 300-800 bp PCR production was isolated using 2% agarose gel electrophoresis. Sequencing was performed on the Illumina Nova Seq 6000 at the Novogene Bioinformatics Institute, Beijing, China.
|
| |
|
| Library strategy |
Bisulfite-Seq |
| Library source |
genomic |
| Library selection |
RANDOM |
| Instrument model |
Illumina NovaSeq 6000 |
| |
|
| Data processing |
For WGBS, trim_galore(v0.6.4) were used to remove low quality reads and adaptor sequence. Reads pass quality control were mapped to mm10 mouse genome using BitMapperBS(v1.0.2.2) in pair-end mode with parameters "--sensitive --pbat --unmapped_out" at first. Unmapped reads were output by samtools(v1.11) and mapped again using BitMapperBS's single-end mode. Aligned bam files were merged and sorted by samtools and the methylation information in MethylKit and bedGraph format were extracted by MethylDackel(v0.4.0) with default parameters. Bed format methylation files were generated by local scripts and BigWig files used in IGV were generated by UCSC binary tools bedGraphToBigWig(v4).
For RNA-seq, trim_galore(v0.6.4) were used to quality control for raw fastq data. After that, clean reads were mapped to mm10 genome by STAR(2.7.2b) with parameters “--quantMode TranscriptomeSAM GeneCounts” to output alignments translated into transcript coordinates in the bam files, and read counts of each genes, which were then used to calculate TPM (Transcript Per Milion).
Genome_build: mm10
Supplementary_files_format_and_content:
bed: Filtered six columns bed format methylation information. Six columns correspond to chromosome, start site, end site, methylation levels (%), counts of C, strand, counts of T. Only CpG site with coverage greater than 3 were remained.
methylkit: methylation call data used for R package methylkit(v1.12.0).
expression levels: raw counts and TPM values of each gene
expression matrix
|
| |
|
| Submission date |
Dec 09, 2020 |
| Last update date |
Apr 23, 2023 |
| Contact name |
Jiansen Lu |
| E-mail(s) |
jiansenlu@pku.edu.cn
|
| Organization name |
Peking University
|
| Department |
Biomedical Pioneering Innovation Center (BIOPIC)
|
| Lab |
Fuchou Tang
|
| Street address |
No. 5 Yiheyuan Road, Haidian District
|
| City |
Beijing |
| ZIP/Postal code |
100871 |
| Country |
China |
| |
|
| Platform ID |
GPL24247 |
| Series (1) |
| GSE162903 |
Base-editor-mediated multiplexed inactivation of DNA methyltransferases reveals essential roles of miRNAs in mouse gastrulation |
|
| Relations |
| BioSample |
SAMN17036748 |
| SRA |
SRX9656634 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4965009_WGBS_DX1_3.combined_CpG.methylKit.txt.gz |
203.1 Mb |
(ftp)(http) |
TXT |
| GSM4965009_WGBS_DX1_3_combined_CpG_3x_flt.bed.gz |
96.0 Mb |
(ftp)(http) |
BED |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
|
|
|
|
 |
