|
| Status |
Public on Jan 06, 2014 |
| Title |
Mettl3 KD at 4h, biological rep2 |
| Sample type |
RNA |
| |
|
| Source name |
mESC Mettl3 knockdown at 4 hour
|
| Organism |
Mus musculus |
| Characteristics |
time: 4 hour
treatment: Mettl3 KD
cell line: J1
|
| Treatment protocol |
ES cells J1 were seeded 2×105 per well in 6-well plate and transfected with siRNAs by lipofectamine RNAiMAX (Invitrogen) following manufacturer’s protocol. After 48 hours cells were reseeded and transfected with siRNA again. After another 48 hours cells were collected for analysis. Target sequences of siRNA: Mettl3, GGACTGCGATGTGATTGTA, GACGAATTATCAATAAGCA; Mettl14, CCGGATGTACAGAGGAAAT, GGGAACTCATCAGACTAAA, GCACCTCGGTCATTTATAT Scramble
|
| Growth protocol |
Mouse ES cells were maintained on feeder layers of irradiated mouse embryonic fibroblasts (MEFs) in DMEM (Invitrogen) supplemented with 15% fetal bovine serum (FBS, Hyclone), 2 mM L-glutamine, 0.1 mM non-essential amino acids (Gibco), 0.1 mM β-mercaptoethanol (Sigma) and 500 units/ml leukemia inhibiting factor (LIF, Chemicon). Lentiviral constructs including shRNA were purchased from Sigma. To produce lentivirus, lentiviral constructs and packaging constructs were transfected into 293ft cells by calcium phosphate reagent (Clontech). About 36 hours after transfection, viral supernatants were collected and supplemented with 6 μg/μl polybrene (Millipore). ES cells were incubated with virus-containing medium for 12 h. 3 days after infection, 2 μg/ml puromycin (Sigma) were added to medium for stable cell lines selection.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted using Trizol reagent (Invitrogen). RNA was treated with RNAse free DNAse I (Roche) to deplete DNA contamination. PolyA RNA was purified using GenElute™ mRNA Miniprep Kit (Sigma Aldrich) per manufacturer’s instruction.
|
| Label |
biotin
|
| Label protocol |
RNA was labeled using the Affymetrix IVT Express kit
|
| |
|
| Hybridization protocol |
RNA was hybridized to Affymetrix GeneChip Mouse Gene 2.0 ST Arrays following manufacturers instructions, by the Ramaciotti Centre for Gene Function, UNSW, Sydney, Australia
|
| Scan protocol |
Arrays were scanned following manufacturers instructions, by the Ramaciotti Centre for Gene Function, UNSW, Sydney, Australia
|
| Description |
Gene expression data from mouse embryos stem cell treated with shRNA knockdown for Mettl3 for 4 hour
|
| Data processing |
Microarray CEL files were imported and normalized using rma function from bioconductor package oligo.
|
| |
|
| Submission date |
May 13, 2013 |
| Last update date |
Oct 20, 2014 |
| Contact name |
Yue Li |
| E-mail(s) |
gorillayue@gmail.com
|
| Organization name |
Massachusetts Institute of Technology
|
| Street address |
32 Vassar Street, 32-D528
|
| City |
Cambridge |
| State/province |
Massachusetts |
| ZIP/Postal code |
02139 |
| Country |
USA |
| |
|
| Platform ID |
GPL16570 |
| Series (2) |
| GSE46879 |
RNA methylation destabilizes developmental regulators in murine embryonic stem cells (MoGene-2) |
| GSE46880 |
RNA methylation destabilizes developmental regulators in murine embryonic stem cells |
|
