|
| Status |
Public on Dec 20, 2012 |
| Title |
12hr-B |
| Sample type |
RNA |
| |
|
| Source name |
KH2 mES
|
| Organism |
Mus musculus |
| Characteristics |
cell line: KH2
treatment: retinoic acid
time: 12 hours
|
| Treatment protocol |
Media was changed 3 hours before passaging them. Media was aspirated and washed twice with PBS. 2 ml of pre warmed Trypsin/EDTA solution were added and placed in incubator at 370 C for 1minute. During this period colonies float off when flicking the plate. Trypsin activity was stopped by adding 5ml of FCS-ES medium to flask. Colonies were dissociated into single cells by pipetting up and down for several times and pelleting the cells by centrifugation at 1000 rpm for 5 minutes. Media was aspirated and the cells were resuspended in appropriate volume of fresh ES cell medium and cells were seeded in 1:6. ratio into fresh T75 flask with feeders. Plate was tilted several times to distribute the cells evenly and placed in the incubator. Next day media was changed. Cells treated for 2, 4 and and 6 hours with RA were supplemented with differentiation media ( DMEM + 10% Serum + NAA+ 0.3uM RA) . After required period of induction, Media was aspirated and washed twice with PBS. 2 ml of pre warmed Trypsin/EDTA solution were added and placed in incubator at 370 C for 1minute. During this, period colonies float off when flicking the plate. Trypsin activity was stopped by adding 5ml of FCS-ES medium to flask. Colonies were dissociated into single cells by pipetting up and down for several times. Additional 15 ml of Differentiation media (without RA) was added and plated into freshly gelatinized plates. For gelatinized plates, Culture plates were treated half an hour before seeding with 0.1% Gelatin. Gelatin was later aspirated just before seeding of cells. After half an hour media was aspirated and centrifuged for 5 min at 1000 rpm. Pellets were dislodged by gentle tapping and 2 ml trizol were added. These tubes were stored at -80 until RNA isolation.
|
| Growth protocol |
One day before of thawing, T25 plates with feeders were prepared. Aseptically the cell suspension was transferred into a sterile centrifuge tube containing several ml of warm FCS-ES media. Cells were pelleted by centrifuging at 1000 rpm for 5 minutes. Supernatant was aspirated and cells were resuspended into required quantity of fresh media. Cells were distributed evenly by tilting plates several times and then were placed in incubator (at 370 C and 5% CO2). Passage/expand of ES cells: Media was changed 3 hours before passaging them. Media was aspirated and washed twice with PBS. 800l of pre warmed Trypsin/EDTA solution were added and placed in incubator at 370 C for 1minute. During this period colonies float off when flicking the plate. Trypsin activity was stopped by adding 5ml of FCS-ES medium to flask. Colonies were dissociated into single cells by pipetting up and down for several times and pelleting the cells by centrifugation at 1000 rpm for 5 minutes. Media was aspirated and the cells were resuspended in appropriate volume of fresh ES cell medium and cells were seeded in 1:6 ratio into T75 flask. Plate was tilted several times to distribute the cells evenly and placed in the incubator.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA for microarray analysis was isolated from ES cells grown on feeder cells. After induction, cells were washed in PBS, trypsinized, and replated onto a fresh gelatinized plate for 30 minutes. Media and unbound cells were aspirated. Adhered cells were resuspended in Trizol, aliquoted and flash frozen until needed. RNA were isolated as described per manufacture’s instruction and cleaned using RNAeasy Kit ( Quiagen). Quality and quantity of RNA were analyzed by Agilent Bioanlayzer 2100.
|
| Label |
biotin
|
| Label protocol |
Biotin labelling was done with 200 ng total RNA with the Ambion MessageAmp III kit.
|
| |
|
| Hybridization protocol |
Following fragmentation, 12.5 ug of aRNA were hybridized for 16 hr at 45C on Affymetrix Mouse Genome 430 2.0 array. GeneChips were washed and stained in the Affymetrix Fluidics Station 450.
|
| Scan protocol |
Arrays were scanned with GeneChip 3000.
|
| Data processing |
Affymetrix Mouse 430 v2 arrays were analyzed in R, version 2.13, using the packages affy (Gautier et al. 2004), version 1.3, and limma (Smyth et al., 2005), version 3.8.3. Normalization was done using rma.
|
| |
|
| Submission date |
Dec 08, 2011 |
| Last update date |
Dec 20, 2012 |
| Contact name |
Julia Zeitlinger |
| E-mail(s) |
jbz@stowers.org
|
| Organization name |
Stowers Institute for Medical Research
|
| Lab |
Zeitlinger Lab
|
| Street address |
1000 E 50th St
|
| City |
Kansas City |
| State/province |
MO |
| ZIP/Postal code |
64110 |
| Country |
USA |
| |
|
| Platform ID |
GPL1261 |
| Series (2) |
| GSE34279 |
Retinoic acid (RA) induction time-course to profile gene expression during mES cell differentiation |
| GSE34304 |
Poised RNA Polymerase II changes over developmental time and prepares genes for future expression |
|
