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| Status |
Public on Dec 26, 2019 |
| Title |
Dnmt3ab_DKO_E65_ExE_B6DBAxPWK_RNAseq_embryo6 |
| Sample type |
SRA |
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| Source name |
E6.5 extraembryonic ectoderm
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| Organism |
Mus musculus |
| Characteristics |
strain/background: B6/DBA x PWK
parental genotype: WT x WT
tissue: E6.5 extraembryonic ectoderm
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| Extracted molecule |
total RNA |
| Extraction protocol |
As all libraries generated in this study involved low-input techniques, samples collected were directly put into lysis buffer for further library construction.
RNA-seq (Sample 1-13):
For low-input RNA-seq libraries, SMARTer Ultra Low Input RNA cDNA preparation kit (Clontech, 643890) was used for reverse transcription and cDNA amplification. cDNA were then fragmented, adaptor-ligated and amplified using Nextera XT DNA Library Preparation Kit (Illumina) according to the manufacturer’s instructions. Single-end 100bp sequencing was performed on a HiSeq 2500 sequencer (Illumina).
Bisulfite-seq (Sample 24-25):
For low-input whole genome bisulfite sequencing (WGBS), the libraries were constructed using the PBAT method (Clark SJ et al., Nature Protocol. 2017 Mar; 12(3):534-547, PMID:28182018).
Bisulfite-seq (reduced representation) (Sample 14-19):
For low-input RRBS, the libraries were prepared as previously described (Shen L et al., Cell Stem Cell. 2014 Oct 2; 15(4):459-471. PMID:25280220).
H3K27me3 and H3K4me3 profiling using CUT&RUN (Sample 20-23, 26-31):
Eed CTR and matKO embryos H3K4me3 and H3K27me3 CUT&RUN libraries were constructed as previously described (Skene and Henikoff, 2017 eLife. 2017; 6: e21856, PMID: 28079019).
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2500 |
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| Description |
Sample 12
Acute depletion of Dnmt3a/3b by 1-cell CRIPSR/Cas9 injection.
mRNA
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| Data processing |
RNA-seq datasets:
Low quality bases and sequencing adaptors of raw RNA-seq reads were trimmed using Trim Galore (version 0.4.5) with parameters “-q 20 –length 20bp”. Reads after trimming were then aligned to the mm9 reference genome using HISAT2 (version 2.1.0) with parameters “--sp 1000,1000 --dta-cufflinks --no-unal”.
Following reads mapping, uniquely aligned reads were retrieved by selecting the alignment records with “NH:i:1” tag and mapping quality greater than 30.
SNPsplit (version 0.3.2) was used separate parental-allele-specific reads and bamCoverage from deepTools (version 3.0.2) was used to generate bigwig tracks with parameters “--skipNonCoveredRegions --binSize 10 --scaleFactor 1”.
Raw read counts for each gene were generated using TEtranscripts (version 1.5.1) and statistical significance [i.e. false discovery rate (FDR)] was computed using the DESeq R package (version 1.34.1) . Only genes with RPKM > 1 in either group, FDR < 0.05 and fold change (FC > 2) were considered as differentially expressed. RPKM values were generated using Cufflinks (version 2.2.1) with default parameters.
WGBS datasets:
WGBS raw reads were trimmed using Trim Galore (version 0.4.5) with parameters “-q 20 –length 35bp -clip-R1 9 –three_prime_clip_R1 9”. Following reads trimming, Bismark (version 0.17.0) was used to align the reads to the mm9 reference genome.
Only uniquely aligned and non-PCR duplicate reads were used for downstream analyses. PCR-duplicates were identified using “MarkDuplicates” from Picard Tools (version 2.8.0). The DNA methylation level for each CpGs was computed as “reads containing methylated C / (reads containing methylated C + reads containing unmethylated C).
Parental-origins of the WGBS reads were determined using SNPsplit (version 0.3.2) and “bedGraphToBigWig” was used to convert bedGraph format to bigwig tracks. The minimum reads coverage for non-allelic and allelic DNA methylation tracks are five and three, respectively.
RRBS datasets:
RRBS reads were trimmed using Trim Galore (version 0.4.5) with parameters “--rrbs --paired -q 20 –length 35bp”. Bismark (version 0.17.0) was used to align the trimmed reads to the mm9 reference genome with default parameters. B6/DBA and PWK SNPs were masked as “N” for the reference assembly.
Uniquely aligned reads were used to compute methylation levels of each CpGs similar to the WGBS datasets. Generation of allelic DNA methylation tracks are the same as the WGBS datasets except that only CpGs with >5 reads coverage were used.
CUT&RUN datasets:
After quality trimming using Trim Galore version (0.4.5), the CUT&RUN reads were aligned to a custom mm9 reference genome (B6/129 and PWK SNPs masked as “N” for H3K4me3 and B6/DBA and PWK SNPs marked as "N" for H3K27me3) using Bowtie2 (version 2.2.9) with the following parameter: “-t -q -N 1 -L 25 --no-unal -I 150 -X 800 --no-mixed --no-discordant”.
After removing non-uniquely mapped reads and PCR duplicates, MACS2 (version 2.1.1) was used to call H3K4me3 and H3K27me3 peaks.
The CUT&RUN bigwig tracks were generated using BamCoverage in deepTools (version 3.0.2).
For allelic H3K27me3 and H3K4me3 enrichment analyses, the parental origins of the reads were determined using SNPsplit (version 0.3.2).
Genome_build: For datasets involved with Dnmt3l and Eed transgenic mouse lines, reference genome was the mm9 (MGSCv37) build with B6/129 and PWK SNPs masked as "N". For Dnmt3a/3b acute depletion experiments, mm9 (MGSCv37) build with B6/DBA and PWK SNPs masked as "N" was used.
Supplementary_files_format_and_content: *.bw: BigWig files that can be uploaded on a genome browser to view methylated cytosine, H3K27me3, H3K4me3, and RNA levels at different genomic locations.
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| Submission date |
Apr 21, 2019 |
| Last update date |
Dec 26, 2019 |
| Contact name |
Zhiyuan Chen |
| E-mail(s) |
Zhiyuan.chen@cchmc.org
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| Organization name |
Cincinnati Children's Hospital Medical Center
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| Street address |
3333 Burnet Avenue
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| City |
Cincinnati |
| State/province |
Ohio |
| ZIP/Postal code |
45229 |
| Country |
USA |
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| Platform ID |
GPL17021 |
| Series (1) |
| GSE130115 |
Allelic histone-to-DNA methylation switch establishes secondary DMR to maintain noncanonical imprinting |
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| Relations |
| BioSample |
SAMN11475245 |
| SRA |
SRX5717153 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM3732316_Dnmt3ab_DKO_E65_ExE_B6DBAxPWK_RNAseq_embryo6_maternal.bw |
10.0 Mb |
(ftp)(http) |
BW |
| GSM3732316_Dnmt3ab_DKO_E65_ExE_B6DBAxPWK_RNAseq_embryo6_paternal.bw |
9.1 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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