|
| Status |
Public on Oct 16, 2023 |
| Title |
16-Cell_77h_53 |
| Sample type |
SRA |
| |
|
| Source name |
16-cell embryo
|
| Organism |
Mus musculus |
| Characteristics |
tissue: 16-cell embryo
genotype: (C57BL/6J x CBA) x DBA/2J
treatment: -
|
| Treatment protocol |
For induction of parthenogenetic embryos, MII-stage oocytes were collected from superovulated females without mating. After removal of cumulus cells, oocytes were treated with 10 mM Sr2+ for 2 hours in Ca2+-free CZB medium and then incubated in KSOM. For generation of IVF-derived zygote, MII oocytes from F1 female mice (C57Bl/6J × CBA) were inseminated with activated spermatozoa obtained from the caudal epididymides of adult DBA/2J male mice. To inhibit both minor and major ZGA, embryos were treated with 0.1mg/mL a-amanitin from 17 hphCG injection until their collection time.
|
| Growth protocol |
Zygotes were collected from the oviduct and cumulus cells were removed upon brief incubation in M2 media containing hyaluronidase. Zygotes were placed in drops of KSOM and cultured at 37 ºC with 5% CO2.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Early stage zygotes were collected and cultured until they reached the S-phase at each developmental stage, based on their time after hCG injection. Embryos were collected at different time points to achieve sampling over the entire S-phase. For the parthenogenetic embryos and IVF-derived zygotes, the timing of S-phase was calculated based on the time after activation and insemination, respectively. Zona pellucida was removed by exposure to acid Tyrode and each blastomere was dissociated by gentle pipetting after trypsin treatment. For the Repli-seq with physically isolated pronuclei, we distinguished maternal and paternal pronucleus based on their size and their relative position to the second polar body and isolated either of them using micromanipulation. The remaining zygote containing a single pronucleus was also collected after removal of polar body so that both pronuclei from the same zygote were further processed for Repli-seq.
Individual blastomeres or pronuclei were placed into 8-strip PCR tubes containing lysis buffer and extracted DNA was fragmented by heat incubation. Fragmented DNA was tagged by the universal primer (5’-TGTGTTGGGTGTGTTTGGKKKKKKKKKKNN-3’) and amplified with WGA primer sets which have individual barcode. Amplified DNA was purified by the QIAquick 96 PCR Purification Kit (QIAGEN) and concentration was determined by the NanoDrop (Thermo Scientific). Equal amount of DNA from each samples (up to 96 samples) were pooled and 1ug of them was ligated with the illumina adaptors using the NEBNext Ultra II DNA Library Prep Kit (NEB). Illumina sequence (NEBNext Multiplex Oligos for Illumina; NEB) were added to the adaptor- ligated samples by the PCR. Clean up and size selection of the PCR product was done using SPRIselect (Beckman Coulter) and the quality of the library was confirmed by 2100 Bioanalyzer with the High Sensitivity DNA Kit (Agilent).
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| |
|
| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina NovaSeq 6000 |
| |
|
| Data processing |
Sequencing reads were aligned to the mm10 genome using bowtie2 (version 2.3.5) with the --local option. Duplicates were marked using samtools (version 1.9)and reads were filtered by keeping only properly paired reads, removing duplicates and selecting those whose mapping quality was higher or equal to 20. Bed files of the read coordinates were generated with bedtools (version 2.29.0) bamtobed command. Using bedtools intersect, read counts were obtained for contiguous 50kb genomic bins.
The window counts were first normalised to RPM and then each bin by its respective average of all samples within the same stage, aiming to correct for the mappability biases intrinsic to the genomic regions. Outlier regions were then masked, specifically the windows of the lower 5th percentile and the upper 1st percentile values. To correct for low mappability, the windows were segmented with the R package copynumber (version 1.28.0) to keep the segments with the highest 95% of values. The data were centred by the mean and scaled by the interquartile range for each cell and smoothed using a median filter with a running window width of 15 windows, followed by a segmentation with the R package copynumber. Finally, using the function normalmixEM from R package mixtools (version 1.2.0), the segmented values were used to fit a mixture model with two components to identify replicated and non-replicated window populations.
Assembly: mm10
Supplementary files format and content: tab-separated txt file with binary replication status
Library strategy: Single-cell Repli-seq
|
| |
|
| Submission date |
Nov 19, 2022 |
| Last update date |
Oct 16, 2023 |
| Contact name |
Tamas Schauer |
| E-mail(s) |
tamas.schauer@helmholtz-munich.de
|
| Organization name |
Helmholtz Zentrum München
|
| Department |
Institute of Epigenetics and Stem Cells
|
| Street address |
Feodor-Lynen-Straße 21
|
| City |
Munich |
| ZIP/Postal code |
81377 |
| Country |
Germany |
| |
|
| Platform ID |
GPL24247 |
| Series (1) |
| GSE218365 |
Genome-wide analysis of replication timing in preimplantation mouse embryos |
|
| Relations |
| BioSample |
SAMN31800170 |
| SRA |
SRX18318159 |
