|
| Status |
Public on Nov 15, 2016 |
| Title |
Mouse_P1 CLiP Hep-i(-)_2 |
| Sample type |
RNA |
| |
|
| Source name |
Mouse CLiPs at P1 cultured for 8 days without hepatic induction
|
| Organism |
Mus musculus |
| Characteristics |
strain: C57BL/6
gender: Female
hepatic induction: no
passages: P1
|
| Treatment protocol |
For hepatic induction, mouse CLiPs were cultured in the presence or absence of oncostatin M and dexamethasone.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was extracted using microRNeasy kit (Qiagen) following the manufacturer's instruction.
|
| Label |
Cy3
|
| Label protocol |
Total RNA was amplified and labeled with Cyanine 3 (Cy3) using Agilent Low Input Quick Amp Labeling Kit, one-color (Agilent Technologies, Palo Alto, CA) following the manufacturer's instructions. Briefly, 100 ng of total RNA was reversed transcribed to double-strand cDNA using a poly dT-T7 promoter primer. Primer, template RNA and quality-control transcripts of known concentration and quality were first denatured at 65 °C for 10 min and incubated for 2 hours at 40 °C with 5X first strand Buffer, 0.1 M DTT, 10 mM dNTP mix, and AffinityScript RNase Block Mix. The AffinityScript enzyme was inactivated at 70 °C for 15 min. cDNA products were then used as templates for in vitro transcription to generate fluorescent cRNA. cDNA products were mixed with a transcription master mix in the presence of T7 RNA polymerase and Cy3 labeled-CTP and incubated at 40 °C for 2 hours. Labeled cRNAs were purified using QIAGEN’s RNeasy mini spin columns and eluted in 30 ul of nuclease-free water. After amplification and labeling, cRNA quantity and cyanine incorporation were determined using a Nanodrop ND-1000 spectrophotometer and an Agilent Bioanalyzer.
|
| |
|
| Hybridization protocol |
For each hybridization, 0.60 ug of Cy3 labeled cRNA were fragmented, and hybridized at 65 °C for 17 hours to an Agilent SurePrint G3 Rat GE 8x60K Microarray (Design ID: 074809).
|
| Scan protocol |
After washing, microarrays were scanned using an Agilent DNA microarray scanner.
|
| Data processing |
Intensity values of each scanned feature were quantified using Agilent feature extraction software version 10.7.3.1, which performs background subtractions. We only used features which were flagged as no errors (Detected flags) and excluded features which were not positive, not significant, not uniform, not above background, saturated and population outliers (Compromised and Not Detected flags). Normalization was performed using Agilent GeneSpring version 12.6.1. (per chip: normalization to 75 percentile shift; per gene: normalization to median of all samples).
|
| |
|
| Submission date |
Oct 03, 2016 |
| Last update date |
Nov 15, 2016 |
| Contact name |
Takeshi Katsuda |
| E-mail(s) |
[email protected]
|
| Phone |
+1-215-746-5559
|
| Organization name |
University of Pennsylvania
|
| Department |
Perelman School of Medicine
|
| Lab |
Gastroenterology Division
|
| Street address |
421 Curie Blvd, BRB II/III Rm531
|
| City |
Philadelphia |
| State/province |
PA |
| ZIP/Postal code |
19104 |
| Country |
USA |
| |
|
| Platform ID |
GPL21163 |
| Series (2) |
| GSE87579 |
Conversion of terminally committed mouse hepatocytes to culturable bipotent progenitor cells |
| GSE87764 |
Chemical induction of liver progenitor cells from mature hepatocytes |
|
