|
| Status |
Public on Jun 29, 2020 |
| Title |
neonatal Cardiomyocytes IP3 |
| Sample type |
SRA |
| |
|
| Source name |
heart
|
| Organism |
Rattus norvegicus |
| Characteristics |
strain: Wistar
age: 2day
cell type: neonatal Cardiomyocytes
treatment: mock
rip antibody: Cpeb4 Antibody (Sigma Aldrich, catalog# HPA038394, lot# A103984)
sample type: Isolated RNA after CPEB4 IP
|
| Treatment protocol |
Immunprecipiation against Cpeb4 overnight from 10million myocyters
|
| Growth protocol |
Ventricular cardiomyocytes from 1 to 2 day old Wistar rats neonatal hearts (NRCMs) were prepared by trypsin digestion and percoll gradient separation. Myocytes were treated with phenylephrine for 24h and Cpeb4 was immunoprecipitated.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
NRVCMs were harvested in polysomal lysis buffer and sonicated for complete lysis. Lysate was incubated with Cpeb4 AB overnight at 4°C, then for 1h at RT with prepared dynabeads sheep anti rabbit IgG. Coprecipiate was washed 3x with wash buffer (polysomal buffer, 10% Triton, DNase I), three times with high salt buffer (polysomal buffer + 1M KCl, 10% Triton, DNase I) and once with wash buffer. RNA was eluted by addition of Trizol and following RNA clean-up. Lexogen Quant-Seq kit was used for library generation following the manufactures instruction. Libraries were multiplexed and sequenced on a NextSeq550.
exogen Quant-Seq kit was used for library generation following the manufactures instruction using 200ng of isolated RNA. RT was performed with 15cycles.
|
| |
|
| Library strategy |
RIP-Seq |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
NextSeq 550 |
| |
|
| Description |
CPEB4 IP eluate
|
| Data processing |
Reads were trimmed with Cutadapt (v2.5) and mapped to rat genome (Rnor_6.0) with STAR (v2.7) and summarized with featureCounts (v1.6.4)
DESeq2 (Love et al., 2014) with IHW (Ignatiadis et al., 2016) for multiple hypothesis correction was used to determine significantly enriched RNAs in IP samples vs corresponding input controls (adjusted p-value < 0.1; log2 fold-change > 0.5).
Genome_build: Rnor_6.0
Supplementary_files_format_and_content: Matrix table with raw gene counts for every gene and every sample
|
| |
|
| Submission date |
Jun 28, 2020 |
| Last update date |
Jun 30, 2020 |
| Contact name |
Mirko Volkers |
| E-mail(s) |
[email protected]
|
| Organization name |
University Hospital Heidelberg
|
| Street address |
INF 410
|
| City |
Heidelberg |
| ZIP/Postal code |
69120 |
| Country |
Germany |
| |
|
| Platform ID |
GPL25029 |
| Series (1) |
| GSE153409 |
Identification of Cpeb4 binders in cardiomyocyte |
|
| Relations |
| BioSample |
SAMN15394433 |
| SRA |
SRX8627142 |
