|
| Status |
Public on Mar 18, 2017 |
| Title |
Hetero_6hr_rep3 |
| Sample type |
SRA |
| |
|
| Source name |
Hetero, 6hr
|
| Organisms |
Homo sapiens; Mus musculus |
| Characteristics |
sample type: mouse ESC and human endothelial cell, fused
time: 6hr
replicate: rep3
|
| Treatment protocol |
Heterokaryon formation and isolation: The cells were collected in conical tubes after neutralization by MEF media containing 10% FBS. The cells were then counted with hemocytometer and 2X105 pluripotent stem cells were taken. The cells were then centrifuged at 200xg for 5 mins at 4oC, the supernatant was removed, and the cell pellet was resuspended in 25 μL ice-cold cell fusion buffer with 2.5 μL ice-cold HVJ-envelope fusagen. The reaction mixture was placed on ice for 5 mins with regular agitation in 2.5 mins apart. After 5 mins, the cells were centrifuged again and the supernatant was discarded and 2 mL cell fusion buffer was added. The pluripotent cells were then plated onto the endothelial cells. The 6-well dish was then centrifuged at 200g for 5 mins at 4oC. After centrifugation the dish was placed into a 37oC incubator to induce cell fusion. Twenty minutes later, the medium was removed and EGM-2 MV medium was added. For the Co-culture Control, the described procedure was the same except HVJ-enveloped fusagen was not added. The heterokaryons (double-positive cells) can be efficiently sorted by FACS. Heterokaryons (GFP+ and CellTracker Red+) were harvested by FACS at 6, 12, 24, 48, and 72 hours post-fusion.
|
| Growth protocol |
Cell preparation: One day before cell fusion, 4X105 endothelial cells were seeded on one well of a 6-well dish. The endothelial cells were confluent on the day of cell fusion. On the same day, 1 h prior to cell fusion, the endothelial cell medium was replaced with fresh EGM-2 MV medium supplemented with 1 μM Cell Tracker Red, then cells were incubated at 37oC in darkness for 30 mins. Human iPSCs or murine ESCs labeled by transduction with retroviruses encoding GFP were rinsed with PBS followed by accutase treatment at 37oC for 5 mins to dissociate the pluripotent stem cells into single cell suspension.
|
| Extracted molecule |
polyA RNA |
| Extraction protocol |
Heterokaryons (GFP+ and CellTracker Red+) were harvested by FACS at 6, 12 and 24 hours post-fusion and prepared for analysis by RNA-seq. Total RNA from heterokaryons were isolated. Human and mouse mRNA transcripts were isolated from the total RNA samples using polyA-based enrichment using oligo-dT magnetic beads. The majority of contaminating ribosomal RNA was eliminated by this approach. The resulting mRNA was fragmented, reverse transcribed to cDNA, ligated to adapters, and subject to brief PCR amplification in preparation of the Illumina library. The integrity and quality of RNA and complementary DNA were monitored using an Agilent Bioanalyzer 2100.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2000 |
| |
|
| Description |
Mus musculus and Homo sapiens fused
|
| Data processing |
RNA-Seq reads were aligned to the mm9 whole genome using tophat v2.0.12 with parameters --mate-std-dev 200 -p 8 -r 203. We use the full set of knownGene downloaded from the UCSC Genome browser (http://genome.ucsc.edu/cgi-bin/hgTables ) as reference genes. RNA-Seq read counts for each gene in each sample was calculated using Cuffdiff function in Cufflinks version 2.2.1. The Cuffdiff also calculates fragment per kilobase per million reads (FPKM) for each gene.
Fragments Per Kilobase of transcript per Million mapped reads were calculated using cuffdiff v2.2.1
Genome_build: mm9
Supplementary_files_format_and_content: [.fpkm_tracking] tab-delimited text files include FPKM values for each Sample
|
| |
|
| Submission date |
Jul 19, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
Alin Tomoiaga |
| E-mail(s) |
satomoiaga@houstonmethodist.org
|
| Organization name |
Houston Methodist Research Institute
|
| Street address |
6670 Bertner Ave
|
| City |
Houston |
| ZIP/Postal code |
77030 |
| Country |
USA |
| |
|
| Platform ID |
GPL16512 |
| Series (1) |
| GSE84558 |
Discovery of Novel Determinants of Endothelial Lineage: Insights from Chimeric Heterokaryons |
|
| Relations |
| BioSample |
SAMN05416278 |
| SRA |
SRX1959991 |
