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| Status |
Public on Oct 18, 2023 |
| Title |
2cell embryo 38 Mutant scBS-seq |
| Sample type |
SRA |
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| Source name |
embryo
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| Organism |
Mus musculus |
| Characteristics |
cell type: embryo
genotype: Padi6MatP620A/+
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| Growth protocol |
For the experiment, males of 8-12 weeks of age and females of 4-8 weeks of age were used. The day before the start of the experiment, the males were separated. On day 1, at 2 pm, intraperitoneal injections of pregnant mare serum gonadotropin (PMSG) were given to the females. After approximately 52 hours, the same females received intraperitoneal injections of human chorionic gonadotropin (hCG). Male was sacrificed approximately 12 hours after the female hCG injection. The cauda epididymis and vasa deferentia were dissected and the spermatozoa were collected in the human tubal fluid medium (HTF) (EmbryoMax® HTF MR-070-D). Spermatozoa were counted and incubated at 37°C within a humidified atmosphere of 5% CO2 in air. Females were sacrificed approximately 13 hours after the hCG injection. Ampullae were placed in hyaluronidase 1mg/ml (H6254-500MG) drops to break down the oocyte-cumulus complex and collect MII oocyte. The oocytes were moved to the HTF medium and a 2x106 sperm/ml concentration of spermatozoa were added. After 4 - 6 hours of incubation at 37°C within a humidified atmosphere of 5% CO2 in air, the fertilised oocytes were washed, to remove the spermatozoa e debris, transferred in KSOM medium and incubated overnight at 37°C within a humidified atmosphere of 5% CO2 in air. The embryos' status was evaluated in the morning and the next 120 hours.
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| Extracted molecule |
polyA RNA |
| Extraction protocol |
MII oocytes and 2-cell embryos were collected as described before. For 2-cell embryos, the zona pellucida was removed with Tyrode’s solution (SIGMA-ALDRICH), the blastomeres were separated and the polar body was discarded. Then single oocytes and blastomeres were picked and put into a lysis buffer (Buffer RLT Plus Qiagen). DNA and RNA from lysate were separated using the G&T protocol (Angermueller et al. 2016). The magnetic beads (MyOne C1, Life Technologies) were washed and then annealed to oligo dTs. These were used to capture polyadenylated mRNA from individual cell lysate. The supernatant containing the DNA was transferred to a new tube and the beads washed three times in 1xFSS buffer (Superscript II, Invitrogen), 10 mM DTT, 0.005% Tween-20 (Sigma) and 0.4 U/μl of RNAsin (Promega) to remove all DNA residue. Each washing solution was added to the DNA tube to maximise recovery.
The scBS-seq libraries were generated as described (Clark et al., 2017). The DNA was purified from the lysated using a 0.9:1 ratio of Ampure XP Beads (Beckman Coulter) and eluted into 10 μl of water. DNA purified from single cells was treated with the EZ Methylation Direct Kit (Zymo) for bisulfite-conversion following the manufacturer's instructions. First-strand synthesis was performed in five rounds. Initially, the bisulfite-treated DNA was mixed with 40 μl first-strand synthesis mastermix (1 × Blue Buffer (Enzymatics), 0.4 mM dNTP mix (Roche), 0.4 μM 6NF oligo (IDT)) and heated to 65 °C for 2 min and cooled on ice, followed by the addition of 50U of Klenow exo- and incubation at 37 °C for 30 minutes after slowly ramping from 4 °C. This process was repeated four more times with additional reaction mixture added each time, and the final round was incubated for 90 minutes at 37 °C. Exonuclease digestion was carried out by adding 20U of exonuclease I (NEB) in a total volume of 100 μl, at 37 °C for 1 h. The resulting samples were purified using AMPure XP beads with a 0.8:1 ratio. The beads were mixed with 50 μl second-strand master mix (1× Blue Buffer (Enzymatics), 0.4 mM dNTP mix (Roche), 0.4 μM 6NF oligo (IDT). The mixture was heated to 98 °C for 1 min and cooled on ice. Then, 50U of Klenow exo- (Enzymatics) were added. The mixture was incubated on a thermocycler at 37 °C for 90 min after slowly ramping from 4 °C. The resulting samples were purified using a 0.8:1 ratio of AMPure XP beads, and libraries were amplified using a 50 μl PCR mastermix (1× KAPA HiFi Readymix, 0.2 μM PE1.0 primer, 0.2 μM iTAG index primer) The amplification process involved a 2 min step at 95 °C, followed by 14 cycles of 80 s at 94 °C, 30 s at 65 °C, and 30 s at 72 °C, with a final extension for 3 min at 72 °C. Finally, the scBS-seq libraries were subjected to purification using a 0.7:1 ratio of AMPure XP beads. After purification, the libraries were eluted in 15 µl of water, pooled together, and sequenced. The sequencing process involved the generation of pools of 48 libraries, which were sequenced on an Illumina HiSeq 2500 platform. On average, the libraries were sequenced to generate 13.0 million paired-end reads with a read-length of 75 bp.
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| Library strategy |
Bisulfite-Seq |
| Library source |
transcriptomic |
| Library selection |
RANDOM |
| Instrument model |
Illumina HiSeq 2500 |
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| Data processing |
A total of 96 scBS-seq libraries were processed for analysis. The first 6 bp containing the N portion of the random primers, adapters and bases called with poor quality (Phred score <20) were removed using Trim Galore version 0.4.4 (Krueger, 2017) in single-end mode. Only the good quality reads were used for the alignment to the Genome Reference Consortium mouse genome build 39 (GRCm39) using Bismark version 0.18.2 (Krueger & Andrews, 2011) with single-end and non-directional mode followed by deduplication and methylation calling using Bismark functions. The MII scBS-seq libraries having a mapping efficiency <10% or fewer than 500,000 CpGs covered were removed (Supp. Table S20-21). To investigate a possible somatic cell DNA contamination in MII oocytes, we compared the global CpG methylation rates with the methylation of X-chromosome CpG islands, removing the samples with X-chromosome CpG island mean methylation > 12 % (Fig. S5a-c; Castillo-Fernandez et al. 2020); there was no difference in the proportion of oocytes excluded on this basis between Padi6P620A/P620A and control. In total, out of 44 MII oocytes, we kept 10 cells for downstream analysis. The embryos scBS-seq libraries having fewer than 100,000 reads were removed. In total, out of 44 embryos, we kept for 29 the downstream analysis (Fig. S5d,e). The methylation profile for each feature was calculated by the Bisulfite methylation pipeline implemented in SeqMonk.
Assembly: mm39
Supplementary files format and content: Bismark coverage file
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| Submission date |
Oct 15, 2023 |
| Last update date |
Oct 18, 2023 |
| Contact name |
Francesco Cecere |
| E-mail(s) |
francesco.cecerengs@gmail.com
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| Phone |
3888903265
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| Organization name |
University of Campania "Luigi Vanvitelli"
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| Department |
DiStABiF
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| Lab |
Riccio
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| Street address |
Via A. Vivaldi, n. 43
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| City |
Caserta |
| State/province |
Caserta |
| ZIP/Postal code |
81100 |
| Country |
Italy |
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| Platform ID |
GPL17021 |
| Series (2) |
| GSE245424 |
A maternal-effect Padi6 variant causes nuclear and cytoplasmic abnormalities in oocytes as well as failure of epigenetic reprogramming and zygotic genome activation in embryos [scBS-seq] |
| GSE245426 |
A maternal-effect Padi6 variant causes nuclear and cytoplasmic abnormalities in oocytes as well 2 as failure of epigenetic reprogramming and zygotic genome activation in embryos |
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| Relations |
| BioSample |
SAMN37821692 |
| SRA |
SRX22095538 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM7842640_lane8369_TCTCACGG_pool2_38_L001_R1_GRCm39_bismark_bt2.deduplicated.bismark.cov.gz |
4.6 Mb |
(ftp)(http) |
COV |
| GSM7842640_lane8369_TCTCACGG_pool2_38_L001_R3_GRCm39_bismark_bt2.deduplicated.bismark.cov.gz |
4.4 Mb |
(ftp)(http) |
COV |
SRA Run Selector |
| Raw data are available in SRA |
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