|
| Status |
Public on May 15, 2015 |
| Title |
HUVEC shPunisher |
| Sample type |
RNA |
| |
|
| Source name |
HUVEC
|
| Organism |
Homo sapiens |
| Characteristics |
shRNA: stable Punisher (scramble) shRNA
|
| Treatment protocol |
Pluripotent stem cells were differentiated as previously described (Kurian et al, 2012). Briefly, undifferentiated human ES/iPS cells were freshly split on to matrigel-coated plates, making sure the sub-colonies were of small size (~300-500 cells /colony). Cells were differentiated using the chemically defined mesodermal induction media (MIM) (DMEM:F12, 15mg/ml stem cell grade BSA (MP biomedicals), 17.5µg/ml Human Insulin, (SAFC bioscience), 275 µg/ml Human holo transferrin (Sigma Aldrich), 20ng/ml bFGF (Hymanzyme), 50ng/ml Human VEGF-165 aa (Humanzyme), 25ng/ml human BMP4 (Stemgent), 450µM monothioglycerol (Sigma Aldrich), 2.25mM each L-Glutamine and Nonn-essential amino acids (Invitrogen).
|
| Growth protocol |
Human ES cells, H1 (WA1, WiCell), (passage 25-40) were cultured in chemically defined hES/hiPS growth media, mTeSR on growth factor reduced matrigel (BD biosciences) coated plates. Briefly, 70-80% confluent hES/hiPS cells were treated with dispase (Invitrogen) for 7 minutes at 37¡C and the colonies were dispersed to small clusters and lifted carefully using a 5ml glass pipette, at a ratio of ~1:6. Human Umbilical Vein Endothelial Cells (HUVEC) were purchased from Promocell and cultivated in EBM medium supplemented with EGM singlequots (Lonza). iPS/ES-derived endothelial cells were cultivated in EGM-2 bullet kit (Lonza). All the endothelial cells were grown on collagen I coated plates (BD biosciences). All cell lines were maintained in an incubator (37¡C, 5% CO2) with media changes every day (hES/hiPS) or every second day (HUVEC).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA from roughly 10x106 cells was isolated using TRIzol reagent (Invitrogen). Intact total RNA samples were treated with DNase1. All the RNA samples met the following RNA quality- OD260/280 = 2-2.2; OD260/230 ³ 2.0; 28S:18S > 1.0, RIN>7.
|
| Label |
biotin
|
| Label protocol |
200 ng of total RNA were amplified using the Ambion WT Expression Kit (Ambion/Applied Biosystems), labeled using the WT Terminal Labeling Kit (Affymetri
|
| |
|
| Hybridization protocol |
Samples were hybridized to Human Gene 1.0 ST for 16 h at 45ºC and 60 rpm in a GeneChip® Hybridization Oven 640
|
| Scan protocol |
Affymetrix GeneChip Scanner 3000 7G
|
| Description |
HUVEC cells with stable Punisher (scramble) shRNA
|
| Data processing |
CEL files were analyzed using the oligo package in R/Bioconductor and normalized using RMA
|
| |
|
| Submission date |
Feb 13, 2014 |
| Last update date |
May 15, 2015 |
| Contact name |
Christopher Benner |
| E-mail(s) |
cbenner@ucsd.edu
|
| Organization name |
University of California, San Diego (UCSD)
|
| Department |
Medicine
|
| Street address |
9500 Gilman Dr. MC 0640
|
| City |
La Jolla |
| State/province |
California |
| ZIP/Postal code |
92093-0640 |
| Country |
USA |
| |
|
| Platform ID |
GPL6244 |
| Series (2) |
| GSE54964 |
Transcriptomic analysis reveals novel long non-coding RNAs critical for vertebrate development [Affymetrix] |
| GSE54969 |
Transcriptomic analysis reveals novel long non-coding RNAs critical for vertebrate development |
|
