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| Status |
Public on Apr 07, 2020 |
| Title |
DNMT3AB_E6.5_Epi |
| Sample type |
SRA |
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| Source name |
proximal Epiblast
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| Organism |
Mus musculus |
| Characteristics |
strain: B6D2F1 maternal strain crossed with B6D2F1 males
tissue: proximal Epiblast
developmental stage: E6.5
genotype: Dnmt3a + Dnmt3b DKO
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| Treatment protocol |
B6D2F1 oocytes were isolated from 5-10 week old female mice ~12h after a hormone priming superovulation regime (5 IU PSMG and 5IU hCG delivered 46 hours afterwards) and stored in 25 ul droplets of pregassed KSOM media with 1/2 amino acids (Millipore) at 37ºC and 5%C02 . Zygotes were generated either by intracytoplasmic sperm injection of decapitated B6/CAST F1 strain sperm (RNA-seq) or natural mating with B6D2F1 males (Bisulfite-seq) and cultured to E3.5 blastoycsts prior to uterine transfer into pseudopregnant CD-1 strain females (~25-35 g). Post-implantation embryos were isolated according to the embryonic day with a 24 hour offset to accommodate developmental delay as a result of uterine transfer (see extract protocol). Experimental samples were additionally injected with a cocktail of 3-4 discrete gRNA's targeted to exons of respective KO gene and Cas9 mRNA synthesized by in vitro transcription at concentrations of 33.3 ng/ul per gRNA and 200 ng/ul for Cas9. gRNA/Cas9 injections occurred within 6-8 hours post fertilization, followed by equivalent culture in KSOM media to the blastocyst stage as the control.
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| Growth protocol |
Preimplantation embryos were cultured in pre-gassed 25 ul KSOM supplemented with 1/2 amino acids (Millipore) under mineral oil at 37ºC and 5% CO2. After ~84 hours post-fertilization, cavitated blastocysts were transferred into the uterine horns of pseudopregnant females in clutches of 10-15 per horn.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Preparation of samples was performed as described in Smith, Chan, et. al, Nature 2014. The decidua of mated female mice were isolated on the morning of E6.5 and the conceptus removed. Then, under a stereomicroscope, the embryo was carefully bisected along the extraembryonic/embryonic axis, removing the ectoplacental cone from the extraembryonic ectoderm when apparent. After separation, Epiblast and extraembryonic ectoderm incubated for 15 min at 4ºC in 0. 5% Trypsin, 2.5% Pancreatin dissolved in PBS and filtered through a 0.2 mm syringe filter and allowed to rest for 5-10 minutes in KSOM at room temperature. Finally, visceral endoderm was removed by drawing the embryo through a narrow, flame drawn glass capillary and only embryo fragments with no apparent contamination were collected. On average, matched ExE and Epiblast from ~10 embryos were collected per assay. Samples were snap frozen in minimal volume prior to extraction and next-generation library synthesis.
Genomic DNA for Whole Genome Bisulfite Sequencing was isolated according to Smith, Chan et. al, Nature, 2012 using a lysis buffer containing 300 ug/mL Proteinase K, followed by phenol:chloroform extraction and ethanol precipitation. Libraries were prepared using the Accel-NGS™ Bisulfite DNA library kit (Swift Biosciences) according to the manufacturers protocol. Final libraries were generated from 8-9 PCR cycles.
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| Library strategy |
Bisulfite-Seq |
| Library source |
genomic |
| Library selection |
RANDOM |
| Instrument model |
Illumina NovaSeq 6000 |
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| Description |
Whole-genome bisulfite sequencing of Dnmt3a KO E6.5 mouse embryonic tissues
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| Data processing |
Raw base call (BCL) files generated by Illumina sequencers were demultiplexed into FASTQ files using 10X Genomics' Singel-Cell Gene Expression Software Cell Ranger ("mkfastq" pipeline, version 1.3.1, default parameters).
For each experiment FASTQ files were aligned, filtered, barcode and UMI counted using 10X Genomics' Singel-Cell Gene Expression Software Cell Ranger ("count pipeline", version 1.3.1, default parameters). The count pipeline was run jointly on multiple sequencing runs of the same experiment. Genomic reference: "refdata-cellranger-mm10-1.2.0", prepared by 10x Genomics.
For experiments with multiple libraries, all libraries were aggregated using 10X Genomics' Singel-Cell Gene Expression Software Cell Ranger ("aggr pipeline", version 1.3.1, default parameters). The output of Cell Ranger "count" was aggregated and normalized to the same sequencing depth and then the gene-barcode matrices were recomputed on the combined data. Sample aggregation was evaluated with normalizing for sequencing depth differences in the libraries. Normalized UMI counts used the 'mapped' normalization mode. Analysis of the data was based on the normalized UMI counts.
Genome_build: mm10
Supplementary_files_format_and_content: For all samples there is one tar gz archive containing the gene-barcode matrices as produced by the Cell Ranger pipeline. They contain the MEX-formatted UMI count matrices of each sample, or if multiple libraries where sequence, the aggregated gene-barcode matrix. Samples names consist of genotype (KO), developmental state (E6.5, E7.5, E8.5, experiment number (1, 2) with respect to genotype and stage and, if aggregated, library number (a, b). 10x Genomics explains their MEX-formatted UMI counts as follows: "The cellranger pipeline generates a gene-barcode matrix... Each matrix is stored in Market Exchange Format (MEX). It also contains TSV files with genes and barcode sequences corresponding to row and column indices, respectively." More information on the formatting can be found at https://support.10xgenomics.com/single-cell-gene-expression/software/pipelines/latest/output/matrices.
Bisulfite-converted reads were quality filtered and adapter trimmed using cutadapt (v2.4), followed by alignment to the mm10 reference genome with bsmap using the following parameter: -v 0.01 -s 16 -w 100 -S 1 -R -u -q 20. Duplicate reads were removed using GATK MarkDuplicates (v4.1.0.0).
Methylation rates were called using mcall using default parameter and subsequently filtered for coverage (min 10 reads).
Genome_build: mm10
Supplementary_files_format_and_content: Supplementary_files_format_and_content: bigWig file of 10X coverage filtered methylation rate BED file (<chr> <start> <end> <methylation rate>)
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| Submission date |
Mar 10, 2020 |
| Last update date |
Apr 08, 2020 |
| Contact name |
Helene Kretzmer |
| E-mail(s) |
kretzmer@molgen.mpg.de
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| Organization name |
Max Planck Institute for Molecular Genetics
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| Department |
Genome Regulation
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| Lab |
Meissner Lab
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| Street address |
Ihnestraße 63
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| City |
Berlin |
| ZIP/Postal code |
14195 |
| Country |
Germany |
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| Platform ID |
GPL24247 |
| Series (1) |
| GSE137337 |
Epigenetic regulator function through mouse gastrulation |
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| Relations |
| BioSample |
SAMN14350518 |
| SRA |
SRX7890102 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4405464_DNMT3AB_E6.5_Epi.bw |
216.8 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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