 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on Jul 29, 2014 |
| Title |
Two-cell Embryo 5 Blastomere 1 |
| Sample type |
SRA |
| |
|
| Source name |
blastomere
|
| Organism |
Mus musculus |
| Characteristics |
strain: C57BL/6
developmental stage: Two-cell Embryo Blastomere
|
| Treatment protocol |
Prior to blastomere collection, the zona pelucida was removed by Tyrode’s acid solution. The 2- and 4-cell embryos were then immersed in Trypsin solution (Invitrogen) with RNAse inhibitor (1 IU/μl, Clontech) and BSA (50μg/μl) for the separation of the blastomeres. The separated blastomeres were snap frozen, and maintained at -80°C until processed. ICM was isolated by immunosurgery, using anti-mouse IgG and complement (Sigma-Aldrich), following previously described procedures (Handyside & Barton, 1977; Harlow & Quinn, 1979), and snap frozen in Trizol reagent (Invitrogen). TE was isolated by exposure of the blastocyst to concanavalin A conjugated to magnetic beads, followed by trypsin treatment for cell dismemberment. The cells were immersed in PBS (RNAse inhibitor 1IU/μl, BSA 50μg/μl). Following magnetic separation of the trophoblast cells, they were snap frozen in Trizol reagent.
|
| Growth protocol |
Adult female mice (C57BL/6) were super-ovulated by intraperitoneal hormone injections with the interval of 48 hours between injections. The hormones administered were either PMSG followed by hCG (5IU/0.1ml, Sigma-Aldrich), or 0.1ml of PG600 (Intervet) administered twice, equivalent to 2.5 IU of hCG and 5IU of PMSG. Following the second hormonal treatment, the females were mated with males (1:1, C57BL/6). Zygotes, 2-cell, 4-cell and blastocysts were collected 26, 48, 52 and 94 hours post hCG or the second injection of PG600.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
The total RNA was extracted from ICM or TE cells with Trizol (Life Technologies) and the supplement of 1μg of Glycoblue (Invitrogen).
The zygotes, blastomeres and the RNA extracted from ICM and TE were used for cDNA amplification using the SMARTer Universal Low Input RNA kit (Clontech). For all samples, the total amplified cDNA was used for library preparation using Nextera XT DNA Sample Prep Kit (Illumina).
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2000 |
| |
|
| Description |
2E5C1
|
| Data processing |
The 100 base long single end reads were aligned to the mouse genome (mm10, Ensembl (Flicek et al., 2013)), using the STAR aligner (STAR_2.3.0e.Linux_x86_64) (Dobin et al., 2013), allowing for up to 4 mismatches.
Only the reads aligned uniquely to one genomic location, and with alignment quality above 10 were retained for following analyses. The duplicate reads were eliminated with Picard tools (v. 1.86) (http://picard.sourceforge.net).
Reads were converted into bigWig format via UCSC genome tools.
FPKM values were obtained with cufflinks-2.2.1 using Ensembl gtf annotation.
Genome_build: mm10
Supplementary_files_format_and_content: bigWig files report only those reads aligned uniquely to one genomic location, and with alignment quality above 10
Supplementary_files_format_and_content: GSE57249_fpkm.txt includes normalized expression values (FPKM).
|
| |
|
| Submission date |
May 02, 2014 |
| Last update date |
May 15, 2019 |
| Contact name |
Fernando H Biase |
| E-mail(s) |
fbiase@vt.edu
|
| Phone |
540-231-9520
|
| Organization name |
Virginia Tech
|
| Department |
School of Animal Sciences
|
| Lab |
Biase lab
|
| Street address |
175 West Campus Drive
|
| City |
Blacksburg |
| State/province |
VA |
| ZIP/Postal code |
24061 |
| Country |
USA |
| |
|
| Platform ID |
GPL13112 |
| Series (1) |
| GSE57249 |
Cell fate inclination within 2-cell and 4-cell mouse embryos revealed by single-cell RNA sequencing |
|
| Relations |
| BioSample |
SAMN02742600 |
| SRA |
SRX530951 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1377876_2E5C1.bigWig |
47.0 Mb |
(ftp)(http) |
BIGWIG |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
| Processed data are available on Series record |
|
|
|
|
 |
