|
| Status |
Public on Jul 10, 2023 |
| Title |
NF-ICM_mphMax_RNA_rep1 |
| Sample type |
SRA |
| |
|
| Source name |
NF-ICM
|
| Organism |
Mus musculus |
| Characteristics |
development stage: ICM
treatment: morpholino targeted at Max
cell type: Fertilized
|
| Treatment protocol |
Isolated zygotes were injected with either morpholino oligonucleotides targeted at Max, Mcrs1 or 25-base random control morpholino. All morpholinos were designed and synthesized in Gene Tools (https://www.gene-tools.com/). Injected embryos were cultured in G-1 PLUS medium at 37℃ under 5% CO2. Knocking-down (KD)-efficiency was examined at morula or blastocyst stage. ICM and TE were collected for Smart-seq2.
|
| Growth protocol |
BDF1 mice were used as oocyte donors, and the cumulus cells were used as nuclear donors. MII oocytes were enucleated in Chatot-Ziomek-Bavister (CZB) medium containing 5 μg/mL cytochalasin B (CB) by Piezo-driven pipette (PrimeT 130 ech) of an Olympus inverted microscope. The donor cells were transferred into enucleated oocytes by direct injection. The reconstructed oocytes were then cultured in CZB medium for 1h before activation treatment. The cloned constructs were activated by 6 h incubation in 1 mM SrCl2 in Ca2+-free CZB and 5 μg/mL cytochalasin B. Reconstructed embryos were thoroughly washed and cultured in G-1 PLUS medium at 37℃ under 5% CO2.
|
| Extracted molecule |
polyA RNA |
| Extraction protocol |
10 blastomeres were used per reaction, and three replicates were performed for each stage. All isolated blastomeres were washed three times in 0.5% BSA in PBS solution to avoid contamination. RNA-seq libraries were generated using the Covaris DNA shearing protocol for Smart-seq sequence library generation. Briefly, RNAs with a polyadenylated tail were captured, reverse transcribed and pre-amplified. DNA was sheared and purified for library construction.
Libraried were generated using the KAPA HyperPrep Kit for the Illumina platform (kk8504), following the manufacturer's instrunctions. Paired-end 150 bp sequencing was performed in NovaSeq (Illumina) platform in Berry Genomics and Novogen.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
| |
|
| Data processing |
Low-quality and the adaptor sequences were trimmed from the reads using cutadapt (v1.16) with parameters: --trim-n -q 25,25 -m 75 -a AGATCGGAAGAGC -A AGATCGGAAGAGC.
All RNA-seq data were mapped to the mm10 reference genome after cutadapt using hisat2 with parameters ‘--data-cufflinks --no-discordant --no-mixed --no-unal’.
The TPM (Transcripts Per Million) of genes were obtained by StringTie.
Signal tracks for each sample were generated using the deepTools bamCoverage function with parameters "--normalizeUsing RPKM --smoothLength 150 --extendReads 150".
Genome_build: mm10
Supplementary_files_format_and_content: *.bw are bigWig files generated using deepTools. *.exp are gene expression files generated by StringTie.
|
| |
|
| Submission date |
Jan 31, 2022 |
| Last update date |
Jul 10, 2023 |
| Contact name |
Qianshu Zhu |
| E-mail(s) |
[email protected]
|
| Organization name |
Tongji University
|
| Department |
School of Life Science and Technology
|
| Street address |
Siping Road
|
| City |
Shanghai |
| State/province |
Shanghai |
| ZIP/Postal code |
270000 |
| Country |
China |
| |
|
| Platform ID |
GPL24247 |
| Series (2) |
| GSE195760 |
Unreprogrammed H3K9me3 prevents minor zygotic genome activation and lineage commitment in SCNT embryos |
| GSE195762 |
Unreprogrammed H3K9me3 prevents minor zygotic genome activation and lineage commitment in SCNT embryos |
|
| Relations |
| BioSample |
SAMN25512635 |
| SRA |
SRX13993301 |
