|
| Status |
Public on Nov 20, 2013 |
| Title |
Hdac6 KD,replicate A |
| Sample type |
RNA |
| |
|
| Source name |
embryonic stem cells, KD replicate A
|
| Organism |
Mus musculus |
| Characteristics |
development stage: E14
treatment: Lentiviral-based shRNA was used for Hdac6 knock down
|
| Treatment protocol |
In one 10 cm2 dish, 1X10^6 cells were infected with 30 ul of concentrated lentivirus. After 12-15 hours, the media was replaced and cells were cultured until 72 hours post-infection.
|
| Growth protocol |
Feeder-free growth on gelatin coated dishes in standatd ES medium: KO DMEM + 10% FBS, glutamine, LIF, non-essential amino acids, beta-mercaptoethanol.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Trizol, followed by Zymo RNA columns.
|
| Label |
Cy3
|
| Label protocol |
Cyanine-3 (Cy3) labeled cRNA was prepared from 5 ug of total RNA using Low Input Quick Amp Labeling Kit protocol (Agilent) with minor modifications. Briefly, cRNA was amplified by in vitro transcription with amino-allyl UTP (3:2 ratio for amino-ally UTP: UTP) overnight at 37°C. Then, cRNA was purified using Zymo RNA purification columns (Zymo, Irvine, CA) and labelled with Cy3 (GE healthcare) at RT for 60 minutes in the dark. The fluorescence intensity of Cy3 was determined by NanoDrop ND-1000 Spectrophotometer.
|
| |
|
| Hybridization protocol |
Fifty picomoles of Cy3-labelled cRNA was fragmented at 60°C for 30 minutes in a reaction volume of 125 ul containing 1x Agilent fragmentation buffer and 2x Agilent blocking agent following the manufacturers instructions. On completion of the fragmentation reaction, 125 ul of 2x Agilent hybridization buffer was added to the fragmentation mixture and hybridized to Agilent 4X44K Whole Mouse Genome Microarrays for 17 hours at 65°C in a rotating Agilent hybridization oven. After hybridization, microarrays were washed 1 minute at room temperature with GE Wash Buffer 1 (Agilent) and 1 minute with 37°C GE Wash buffer 2 (Agilent), then dried immediately by brief centrifugation.
|
| Scan protocol |
Slides were scanned on the Agilent scanner (G2565CA) immediately after washing.
|
| Description |
gene expression in indicated KD
|
| Data processing |
The scanned images were analyzed with Feature Extraction Software 10.7.1.1 (Agilent) using default parameters (protocol GE1-107 and Grid: 026655_D_F_20120201) to obtain background subtracted and spatially detrended Processed Signal intensities. We used the quantile normalization in R (bioconductor) to obtain the value in the Sample data tables.
|
| |
|
| Submission date |
Nov 09, 2012 |
| Last update date |
Nov 20, 2013 |
| Contact name |
Benson Chen |
| E-mail(s) |
b5chen@ucsd.edu
|
| Organization name |
UC San Diego
|
| Department |
Cellular and Molecular Medicine
|
| Lab |
Bing Ren
|
| Street address |
9500 Gilman Dr. CMM East, Room 2071
|
| City |
La Jolla |
| State/province |
CA |
| ZIP/Postal code |
92093-0653 |
| Country |
USA |
| |
|
| Platform ID |
GPL10333 |
| Series (2) |
| GSE42208 |
Hdac6 regulates Tip60-p400 function in stem cells (gene expression) |
| GSE42329 |
Hdac6 regulates Tip60-p400 function in stem cells |
|
