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| Status |
Public on Nov 12, 2021 |
| Title |
59_I_epi_CastxBdf1 |
| Sample type |
SRA |
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| Source name |
Epiblast
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| Organism |
Mus musculus |
| Characteristics |
tissue: Epiblast
age: E6.5
background: CastxBdf1
genotype: WT
Sex: F
embryo: embryo4
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| Growth protocol |
Embryos were generated as previously described (Wang et al., 2013). Briefly, BDF1 strain female mice (age 6–8 weeks, Jackson Labs) were superovulated by serial Pregnant Mare Serum Gonadotropin (5 IU per mouse, Prospec Protein Specialists) and human chorionic gonadotropin (5 IU, Millipore) injections 46 h apart. The following day, MII stage oocytes were isolated in M2 media supplemented with hyaluronidase (Millipore) and stored in 25 μl drops of pre-gassed KSOM with half-strength concentration of amino acids (Millipore) under mineral oil (Irvine Scientific). Zygotes were generated by piezo-actuated intracytoplasmic sperm injection (ICSI. See (Grosswendt et al., 2020)) using thawed CAST strain sperm in batches of 30–50 oocytes and standard micromanipulation equipment, including a Hamilton Thorne XY Infrared laser, Eppendorf Transferman NK2 and Patchman NP2 micromanipulators, and a Nikon Ti-U inverted microscope. Zygotes for whole genome bisulfite sequencing were generated by natural mating with BDF1 males. For the reciprocal cross, BDF1 males were naturally mated to CAST females and screened for copulation plugs, after which E6.5 stage embryos were isolated accordingly, with the date of the copulation plug score day E0.5. For zygotic disruption, pronuclear stage 3 (PN3) zygotes were injected with 200 ng μl−1 Cas9 mRNA and a 100 ng μl−1 equimolar ratio of 3–4 sgRNAs targeting different exons of an epigenetic regulator gene locus (designed using ChopChop (Labun et al., 2016) and the IDT CRISPR–Cas9 guide RNA checker, as previously described in (Smith et al., 2017)). We designed the gRNA to avoid CAST SNPs to ensure that the gRNAs are equally effective between the alleles. Injections utilized the same microinjection setup and Piezo-actuated injection of front-filled 6-7 μm injection needles. At around 84 h after fertilization, cavitated blastocysts were transferred into the uterine horns of pseudopregnant CD-1 strain females (25–35g, Charles River) generated by mating with vasectomized SW strain males (Taconic), which results in a 24 h offset in gestational time to accommodate implantation, after which animals were monitored for 5 days for embryo isolation at E6.5.
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| Extracted molecule |
total RNA |
| Extraction protocol |
At E6.5, animals were euthanized and the uterine horn removed. Purified epiblast and ExE were isolated according to Ref. (Chenoweth and Tesar, 2010) with a few modifications. Briefly, E6.5 embryos were removed from deciduae and transferred to independent 25 μL drops of M2 media. Using a glass flame-pulled capillary, Reichart’s membrane was reflected, and the embryo carefully bisected along the epiblast, ExE boundary. Then, each Epiblast/ExE pair was transferred into an individual drop of dissociation medium containing 0.5% trypsin and 2.5% pancreatin in PBS (w/v, Sigma). Embryos were cultured with slow orbital rotation at 4ºC for 15 minutes, after which they were transferred into new M2 drops. After ~5 minutes of resting, Epiblast and ExE were passed through a slightly narrower flame-pulled glass capillary to remove the visceral endoderm without disrupting the target tissue. Finally, each tissue was serially washed in 0.1% BSA in PBS prior to snap freezing in lysis buffer.
Approximately 300-600 cells were collected from E6.5 embryos and directly transferred to 2.6 µl of Lysis Buffer (Takara Bio USA, Inc.) followed by snap-freezing at -80°C in preparation for cDNA synthesis using the SMART-Seq v4 assay. Full-length cDNA was prepared using the SMART-Seq v4 Ultra Low Input RNA Kit for Sequencing and sequencing libraries prepared using the Nextera XT DNA library preparation kit (Illumina). The resulting libraries were evaluated using a 4200 TapeStation (Agilent Technologies) and quantified by qPCR. Libraries were pooled and sequenced on an Illumina NovaSeq SP or S1 flow cell using paired-end, 50 bp reads. The E6.5 ExE ∆G9a-Glp sample was isolated and processed into WGBS libraries using the Accel-NGS Methyl-seq kit as described in (Grosswendt et al., 2020) and sequenced on an Illumina NovaSeq using paired-end, 150 bp reads.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
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| Data processing |
The RNA-seq data were aligned to the mm10 reference genome using the STAR aligner (Dobin, A. et al. 2013, Bioinformatics, STAR version 2.5.0 c: –outFilterMultimapNmax 1). The read counts for every RefSeq isoform (RefSeq gene annotation (downloaded February 2018) were determined using the htseq-count phyton script (Anders, S. et al. 2015 Bioinformatics, version 0.6.1) and further normalized in R. The Allelome.PRO approach was used to calculate allele-specific expression from the RNA-seq data as outlined in Andergassen D. et al. 2015 NAR. Briefly, the Allelome.PRO pipeline uses strain-specific single-nucleotide polymorphisms (SNPs) to assign RNA-seq reads to the corresponding allele in F1 crosses.
Genome_build: mm10
Supplementary_files_format_and_content: bigwig
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| Submission date |
Mar 30, 2021 |
| Last update date |
Nov 12, 2021 |
| Contact name |
Daniel Andergassen |
| E-mail(s) |
daniel.andergassen@tum.de
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| Organization name |
Technical University Munich
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| Lab |
Andergassen
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| Street address |
Biedersteiner Straße 29
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| City |
Munich |
| ZIP/Postal code |
80802 |
| Country |
Germany |
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| Platform ID |
GPL24247 |
| Series (1) |
| GSE171206 |
Diverse epigenetic mechanisms maintain parental imprints within the embryonic and extraembryonic lineages |
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| Relations |
| BioSample |
SAMN18544919 |
| SRA |
SRX10484991 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM5220750_59_I_epi_CastxBdf1.bw |
163.1 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
| Processed data are available on Series record |
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