|
| Status |
Public on Oct 25, 2021 |
| Title |
MFA_shC-89 |
| Sample type |
SRA |
| |
|
| Source name |
embryo
|
| Organism |
Macaca fascicularis |
| Characteristics |
day: embryo invitro day 7
shRNA: shscramble
embryotag: embryo3
celltype: TrB
|
| Growth protocol |
Oocytes were collected by laparoscopic follicular aspiration approximately 32–35 h after rhCG administration. Follicular contents were added to HEPES-buffered Tyrode’s albumin lactate pyruvate (TALP) medium containing 0.3% bovine serum albumin (BSA) (Sigma-Aldrich, USA) at 37°C. Oocytes were stripped of cumulus cells by pipetting after a brief exposure (< 1 min) to hyaluronidase (0.5 mg/mL) in TALP-HEPES to visually select nuclear-based mature metaphase II (MII; first polar body present) oocytes. The mature oocytes were immediately subjected to intracytoplasmic sperm injection and then cultured in Connaught Medical Research Laboratories (CMRL) -1066 media (Thermo Fisher Scientific, USA) containing 10\% fetal bovine serum (FBS, Gibco, USA) at 37°C in 5% CO2. Fertilization was confirmed by the presence of a second polar body and two pronuclei. Zygotes were then cultured in chemically defined hamster embryo culture medium-9 containing 10% FBS at 37°C in 5% CO2 to allow embryo development. The culture medium was replaced every other day until the blastocyst stage.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Isolation of single cells. The embryos were washed in PBS for 3 times, washed in 0.25% trypsin (T4799; SigmaAldrich) 2 times, incubated with 0.25% trypsin for around 15 min at 37 °C, and terminated by DFBS. Embryo was dissociated into single cells by repeated pipetting and dispersed in 1% DFBS/PBS. A single cell was pipetted into a PCR tube. All operations above were performed under the Nikon SMZ645 microscopy.
The synthesis and amplification of full-length cDNAs were performed following Smart-seq2 protocol4. Briefly, single cells were washed in DPBS (Gibco 14190-144) and picked into lysis buffer using Pasteur pipettes under a dissecting microscope. Reversetranscription reactions and pre-amplification were performed using SuperScript II (Invitrogen 18064-014) and KAPA HiFi HotStart ReadyMix (KAPA Biosystems KK2601), respectively. The quality of the cDNAs were evaluated by Bioanalyzer 2100. Library construction and sequencing were performed by Annoroad Gene Technology (http://www.annoroad.com/). Pair-end sequencing was performed on Illumina X-ten platform.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
HiSeq X Ten |
| |
|
| Data processing |
The sequencing qualities of 166 scRNA-seq profiles were examined with the FASTQC.
The scRNA-seq data were aligned to Macaca fascicularis genome (macFas5) with HISAT2 (v 2.1.0).
The alignment results of HISAT2 in the SAM format were converted to BAM format and sorted with SAMTools (v 1.1).
Quantified transposable element (TEs) and gene expression of single-cell sequencing data using scTE software.And annoted wtih http://ftp.ensembl.org/pub/release-102/gtf/macaca_fascicularis/Macaca_fascicularis.Macaca_fascicularis_5.0.102.gtf.gz http://hgdownload.soe.ucsc.edu/goldenPath/macFas5/database/rmsk.txt.gz
Single cell dimensionality reduction and visualization use R package seurat (v4.0.1)
Genome_build: macFas5
Supplementary_files_format_and_content: transposable element (TEs) and gene expression (Counts) of single-cell sequencing data were stored in seurat object cymonkeyshRNAdim15r3dim15TrBEPIPrE.rds.
|
| |
|
| Submission date |
Aug 13, 2021 |
| Last update date |
Oct 25, 2021 |
| Contact name |
Tianqing Li |
| Organization name |
Kunming University of Science and Technology
|
| Department |
Yunnan Key Laboratory of Primate Biomedical Research & Institute of Primate Translational Medicine
|
| Street address |
727 South Jingming Road, Chenggong District
|
| City |
Kunming |
| State/province |
Yunnan |
| ZIP/Postal code |
650500 |
| Country |
China |
| |
|
| Platform ID |
GPL23096 |
| Series (1) |
| GSE182061 |
The Long Terminal Repeats of ERV6 Are Activated in Pre-Implantation Embryos of Cynomolgus Monkey |
|
| Relations |
| BioSample |
SAMN20771378 |
| SRA |
SRX11742702 |
