 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on Oct 18, 2023 |
| Title |
MII oocyte 3 wild-type scRNAseq |
| Sample type |
SRA |
| |
|
| Source name |
oocyte
|
| Organism |
Mus musculus |
| Characteristics |
cell type: oocyte
genotype: Padi6+/+
|
| 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.
|
| 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.
mRNA on the beads was processed further for cDNA conversion. This involved the resuspension in 10 μl of reverse transcriptase mastermix (100 U SuperScript II (Invitrogen), 10 U RNAsin (Promega), 1 × Superscript II First-Strand Buffer, 5 mM DTT (Invitrogen), 1 M betaine (Sigma), 9 mM MgCl2 (Invitrogen), 1 μM Template-Switching Oligo (TSO, Eurogentec), 1 mM dNTP mix (Roche). The mRNA mixture was reverse transcribed by incubation for 60 min at 42 °C followed by 30 min at 50 °C and 10 min at 60 °C. The cDNA obtained was subjected to PCR amplification by adding 11 μl of 2x KAPA HiFi HotStart ReadyMix and 1 μl of ISPCR primer (2 μM). The amplification was carried out in a thermocycler at 98 °C for 3 min, followed by 15 cycles of 98 °C for 15 s, 67 °C for 20 s, 72 °C for 6 min and the final extension step at 72 °C for 5 min. The amplified product was purified using Ampure XP beads with a 1:1 ratio and eluted into 20 μl of water. Libraries were prepared from 100 to 400 pg of cDNA using the Nextera XT Kit (Illumina), per the manufacturer's instructions but with one-fifth volumes. All 96 single-cell RNA-seq libraries were pooled together and sequenced on the Illumina NextSeq platform to an average depth of 4.2 million reads, using paired-end 75 bp read-length settings.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic single cell |
| Library selection |
cDNA |
| Instrument model |
NextSeq 550 |
| |
|
| Description |
3_MII_wt
|
| Data processing |
A total of 96 libraries were adapted and quality trimmed (Phred score <20) using Trim Galore version 0.4.4 (Krueger, 2017). We also sequenced 4 negative controls to archive technical problems. The good quality reads were aligned to the Genome Reference Consortium mouse genome build 39 (GRCm39) with HiSat2 version 2.1.0 (Kim et al., 2015) in paired-end mode. The data were quantified in SeqMonk and analysed in Rstudio. We exported the raw count matrix by SeqMonk andwe calculate the differential expression following the DESeq2 pipeline (Love et al, 2014).
Assembly: mm39
Supplementary files format and content: raw count expression matrix
|
| |
|
| Submission date |
Oct 15, 2023 |
| Last update date |
Oct 18, 2023 |
| Contact name |
Francesco Cecere |
| E-mail(s) |
francesco.cecerengs@gmail.com
|
| Phone |
3888903265
|
| Organization name |
University of Campania "Luigi Vanvitelli"
|
| Department |
DiStABiF
|
| Lab |
Riccio
|
| Street address |
Via A. Vivaldi, n. 43
|
| City |
Caserta |
| State/province |
Caserta |
| ZIP/Postal code |
81100 |
| Country |
Italy |
| |
|
| Platform ID |
GPL21626 |
| Series (2) |
| GSE245423 |
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 [scRNA-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 |
|
| Relations |
| BioSample |
SAMN37821890 |
| SRA |
SRX22095563 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
|
|
|
|
 |
