|
| Status |
Public on May 13, 2013 |
| Title |
RNA_C2C12_6hDelta_repl2 |
| Sample type |
SRA |
| |
|
| Source name |
C2C12, Delta 6h
|
| Organism |
Mus musculus |
| Characteristics |
cell line: C2C12
treatment: Delta 6h
technique: cDNA-sequencing
|
| Treatment protocol |
Delta treatments: For ligand-dependent activation of Notch signaling, cells were grown in the presence of the extracellular domain of Delta-like 1 fused to the Fc fragment of human IgG (Hicks et al., 2002) (kindly provided by N. Gupta upon permission from G. Weinmaster). Culture dishes were first coated with 10 µg/ml anti-Fc antibody for 1h at RT. Dishes were then incubated with conditioned medium from transfected 293T cells expressing the fusion proteins, for 1h at RT. C2C12 cells were plated in fresh medium and grown for 6h or 24h on the immobilized-ligand containing dishes.
|
| Growth protocol |
C2C12 cells were cultured in DMEM medium supplemented with 20% fetal bovine serum and 1% penicillin/streptomycin (Invitrogen) at 37 °C in 5% CO2 atmosphere.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Strand-specific RNA-seq using RiboZero: Total RNA was extracted using the RNeasy Mini and Micro kit (Qiagen) following the manufacturer's instructions. Ribosomal RNA was removed from 200 ng total RNA using the Ribo-Zero rRNA Removal Kit Low Input for Human/Mouse/Rat (Epicentre Biotechnologies) according to the manufacturer’s protocol. RNA was subsequently fragmented in fragmentation buffer (40 mM Tris-Ac pH8.2, 100 mM KAc, 30 mM MgAc) for 90 s at 95 ºC and purified by ethanol purification. cDNA was synthesized using random hexamers by Superscript III Reverse Transcriptase (Invitrogen) in the presence of 6 ng/µl ActinomycinD. cDNA was purified (MinElute Reaction Cleanup Kit, QIAGEN) and subjected to second strand synthesis by E. coli DNA Polymerase I (Invitrogen) and E. coli DNA Ligase (New England Biolabs) in the presence of RNaseH (Ambion). For second strand synthesis, dUNTPs were used, containing dUTP instead of dTTP. Ds-cDNA was purified (MinElute Reaction Cleanup Kit, QIAGEN) and prepared for Illumina sequencing according to standard procedures using the DNA sample Prep Kit (Illumina, cat# IP-102–1001) and starting from the A-tailing step. After ligation of adapters, a 200 bp band is excised using the E-Gel system (Invitrogen). Before final PCR amplification, uracil-containing second strand DNA was removed by USER enzyme (New England Biolabs) for 15 min at 37 ºC followed by 10 min at 95 ºC in 1x Phusion Buffer (Finnzymes). PCR amplification was performed with 14 cycles.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina Genome Analyzer IIx |
| |
|
| Description |
Mouse myoblast cell line (established from thigh muscle from an adult C3H strain female).
Ribozero processed and fragmented RNA.
|
| Data processing |
Strand-specific RNA-seq: Cluster generation and sequencing-by-synthesis (36 bp) was performed using the Illumina Genome Analyzer IIx (GAIIx) platform according to standard protocols of the manufacturer (Illumina). The image files generated by the Genome Analyzer were processed to extract DNA sequence data. Sequences were aligned to the mouse (mm9) reference genome using the Illumina Analysis Pipeline, allowing one mismatch. Only the tags uniquely aligning to the genome were considered for further analysis. Further analysis was performed using the 36 bp sequence reads. The output data were converted to Browser Extensible Data (BED) files for downstream analysis and Wiggle (WIG) files for viewing the data in the UCSC Genome Browser. All sequence analyses were conducted based on the Mus musculus mm9 genome assembly.
Genome_build: mm9
|
| |
|
| Submission date |
Apr 11, 2012 |
| Last update date |
May 15, 2019 |
| Contact name |
Arjen Brinkman |
| E-mail(s) |
arjen.brinkman@gmail.com
|
| Organization name |
Radboud University, Nijmegen Center for Molecular Life Sciences
|
| Department |
Molecular Biology
|
| Lab |
Stunnenberg
|
| Street address |
NCMLS #274, Geert Grooteplein Zuid 30
|
| City |
Nijmegen |
| ZIP/Postal code |
6525 GA |
| Country |
Netherlands |
| |
|
| Platform ID |
GPL11002 |
| Series (1) |
| GSE37184 |
Dynamic binding of RBPJ is determined by Notch signalling status |
|
| Relations |
| SRA |
SRX140350 |
| BioSample |
SAMN00854810 |
