|
| Status |
Public on Jul 17, 2017 |
| Title |
IP HA_replicate |
| Sample type |
RNA |
| |
|
| Source name |
Control IP of HA-Msi1
|
| Organism |
Xenopus laevis |
| Characteristics |
tissue: oocytes
antibody: Anti -HA (Roche 1867431)
|
| Treatment protocol |
Stage VI oocytes were lysed and IPs were performed as described above with the indicated antibody. Co-Immunoprecipitated RNAs were eluted in proteinase K buffer (200 mM Tris pH7.5, 100M NaCl, 10 mM EDTA, 1%SDS) with 100 µg of proteinase K (30 min at 37ºC).
|
| Growth protocol |
α IPs and western blot were performed with αCPEB1 rabbit polyclonal antibodies21, αHA (Roche, 1867431). For IP, antibodies were covalently cross-linked to protein A-Dynabeads or protein G dynabeads (Invitrogen). 25 oocytes per condition were lysed (20 mM Tris–HCl pH 8.0, 1 mM EDTA, 0.5% NP-40, 1 mM MgCl2, 100 mM NaCl) and incubated with RNAse A, when indicated, for 20 min at 37°C (100 μg.mL-1). Oocyte extracts were precleared with 25 μL dynabeads for 1 h at 4ºC, then incubated for 2 h at 4ºC with the antibody crosslinked to the beads.
|
| Extracted molecule |
cytoplasmic RNA |
| Extraction protocol |
RNA was then extracted by phenol chloroform followed by isopropanol precipitation.
|
| Label |
biotin
|
| Label protocol |
Amplified cDNA was purified and quantified on a Nanodrop ND-1000 spectrophotometer (Thermo-Fischer). 8 µg cDNA was subsequently fragmented by DNAseI and biotinylated by terminal transferase obtained from GeneChip Mapping 250K Nsp Assay Kit (Affymetrix).
|
| |
|
| Hybridization protocol |
Hybridization, washing, staining and scanning of Affymetrix GeneChip Xenopus laevis Genome 2.0 arrays were performed following the manufacturer’s recommendations
|
| Scan protocol |
Scanned images (DAT files) were transformed into intensities (CEL files) by AGCC software (Affymetrix).
|
| Description |
Control IP of HA-Msi1 (no HA-msi2 overexpression
|
| Data processing |
GeneChip Xenopus laevis Genome 2.0 Array were processed with Bioconductor, using RMA background correction, quantile normalization and RMA summarization. Significantly enriched target genes in CPEB1 vs. preserum and HA-MSI vs. HA samples were detected with the phenoTest package, using a fold-change threshold of 1.5 times or more and a Benjamini-Hogberg adjusted p-value < 0.05.
|
| |
|
| Submission date |
Apr 21, 2015 |
| Last update date |
Jul 17, 2017 |
| Contact name |
Oscar Reina Garcia |
| E-mail(s) |
oscar.reina@irbbarcelona.org
|
| Organization name |
IRB Barcelona
|
| Department |
Biostatistics and Bioinformatics
|
| Street address |
C/Baldiri Reixac 10
|
| City |
Barcelona |
| State/province |
Barcelona |
| ZIP/Postal code |
08028 |
| Country |
Spain |
| |
|
| Platform ID |
GPL10756 |
| Series (2) |
| GSE68095 |
Msi1 assists CPEB1-driven polyadenylation [CPEB1 & Msi1 RIP replicate] |
| GSE68099 |
Msi1 assists CPEB1-driven polyadenylation |
|
