 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on Jan 01, 2018 |
| Title |
8-cell_embryo1_8b |
| Sample type |
SRA |
| |
|
| Source name |
8-cell_embryo1_8
|
| Organism |
Mus musculus |
| Characteristics |
strain: ICR
tissue/cell type: mouse 8-cell embryo
|
| Growth protocol |
Isolation of zygotes, 2-cell, 4-cell, 8-cell embryos, inner cell mass (ICM) and trophectoderm (TE). 6- to 8-weeks old ICR female mice were injected with 7.5IU human chorionic gonadotropin (hCG) 46~48h after injection of 7.5IU pregnant mare serum gonadotrophin (PMSG). 2-cell, 4-cell and 8-cell stage embryos were collected from the oviduct of mated mice at 48, 56 and 70h post-hCG, respectively. The embryos were treated with Acid Tyrode’s solution to remove the zona pellucida and then were transferred to trypsin-EDTA and incubated at 37°C for 5 minutes. Then the embryos were washed with PBS-BSA buffer gently to dissociate the embryos into individual blastomeres. ICM and TE were isolated from mouse E3.5 blastocysts (Yan et al. 2013). The trophectoderm was isolated from the blastocysts with zona pellucida removed under a microscope by mechanically cut using a 30G needle. The exposed ICM was gently scraped from the trophectoderm. These two types of cells were then treated with Accutase or 0.005% trypsin at 37 °C for dissociated into single cell. Cell lines. Mouse embryonic fibroblast (MEF) was derived from E13.5 embryos of ICR strain. Mouse embryonic stem cells (mESCs) were cultured on 0.1% gelatinized tissue culture plates and were passaged every two days.
|
| Extracted molecule |
polyA RNA |
| Extraction protocol |
The RNA of single mESC was extracted by the MAST-Seq protocol which was developed from the single cell RNA-Seq technique we previously developed (Tang et al. 2010b).
For the library constructed for Illumina sequencing platform, 10-100ng amplified cDNA was fragmentized to about 200bp fragments by Covaris S2, and purified with DNA Clean & Concentrator™-5. Then NEBNext® Ultra™ DNA Library Prep Kit for Illumina® (NEB, Catalog #E7370L) was used to construct the cDNA library. The cDNA libraries were sequenced on Illumina HiSeq 2000 and 2500 instrument with 100bp reads and pair-end parameter. For the libraries constructed for Thermo Fisher Proton sequence platform, First, 10-100ng amplified cDNA was fragmentized to about 250bp fragments by Covaris S2, and purified with DNA Clean & Concentrator™-5. The Ion XpressTM Plus Fragment Library Kit (ThermoFisher, cat. no. 4471269) and Ion XpressTM Barcode Adapters 1-16 Kit (Thermo Fisher, cat. No. 4471250) were used to construct the library. Then, sequencing was performed using PI chips with the Ion PI Sequencing 200 kit v3 on Ion PI Proton system.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Ion Torrent Proton |
| |
|
| Description |
Sequenced on Thermo Fisher Proton
Sample 124
|
| Data processing |
library strategy: MAST-seq (Measure a single cell twice by RNA-Seq)
For data from Illumina, Illumina CASAVA version 1.8 were used to the basecalling. For data from Ion Proton, base calls were collected using Ion Torrent Suite v4.3 software.
Adaptor contamination and low-quality reads were discarded from the raw data.
For Illumina paired-end reads, TopHat (version 2.0.10) were used for sequence alignment, and FPKM values were generated by Cufflinks (version 2.1.1). For Ion Proton RNA-Seq reads, 2-step alignment method which combined STAR (version 2.3.0) and bowtie2 (version 2.1.0) was applied. Unmapped reads produced by STAR were re-mapped with bowtie2 in the “very-sensitive-local” mode, and merged bam files were taken as input by Cufflinks.
Genome_build: Reference sequence and transcript annotation files were from UCSC genome assembly mm10.
Supplementary_files_format_and_content: FPKM table generated by cufflinks
|
| |
|
| Submission date |
Jan 26, 2015 |
| Last update date |
May 15, 2019 |
| Contact name |
YangLu Yang |
| E-mail(s) |
lucyyang1991@hotmail.com
|
| Organization name |
Beijing Friendship Hospital, Capital Medical University
|
| Street address |
Beijing Xicheng District Yongan Road NO.95
|
| City |
Beijing |
| State/province |
Beijing |
| ZIP/Postal code |
100053 |
| Country |
China |
| |
|
| Platform ID |
GPL18635 |
| Series (1) |
| GSE65160 |
Authentic single cell RNA-Seq by measuring the transcriptome of an individual cell twice |
|
| Relations |
| BioSample |
SAMN03294994 |
| SRA |
SRX852799 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1591905_8-cell_embryo1_8b.genes.fpkm_tracking.gz |
672.9 Kb |
(ftp)(http) |
FPKM_TRACKING |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
|
|
|
|
 |
