|
| Status |
Public on Jun 11, 2021 |
| Title |
BeWo HA-GCM1 IP DNA rep2 |
| Sample type |
genomic |
| |
|
| Source name |
BeWo 50 mM forskolin, 24hr HA-bead IP 2
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: BeWo cells stably expressing HA-GCM1 (BeWo33)
treatment: 50 mM forskolin, 24hr
chip antibody: Monoclonal Anti-HA-Agarose (Sigma, A2095)
|
| Treatment protocol |
BeWo31 was treated 50mM forskolin for 24 hours.
|
| Growth protocol |
BeWo31 cell was cultured in 15% FBS F12K medium containing 1X Penicillin-Streptomycin-Glutamine.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cells were treated with 4% formaldehyde to crosslink DNA and proteins. Lysates were sonicated and GCM1-DNA complexes were isolated with antibody.
|
| Label |
biotin
|
| Label protocol |
DNA fragments were pulled down by sequential chromatin immunoprecipitation. Amplification of dUTP-incorporated DNA was performed by ligand-mediated PCR (LMPCR). Libraries were prepared according to Affymetrix instructions (Chromatin Immunoprecipitation Assay Protocol). Amplified ChIP targets were cut with Uracil DNA Glycosylase (UDG) and APE1 restriction enzyme treatment into short fragments (~70-80bp). The samples were to be used for fragmentation analysis using a Bioanalyzer. Finally, DNA fragments were labeled by Terminal Deoxynucleotidyl Transferase and ready for chip hybridization.
|
| |
|
| Hybridization protocol |
The labeled DNA was hybridized to the Affymetrix GeneChip human promoter arrays for 16hr at 45 C with constant rotation (60 rpm). Affymetrix Fluidics Station 450 was used to wash and stain the Chips, removing the non-hybridized target and incubating with a streptavidin-phycoerythrin conjugate to stain the biotinylated cDNA. The staining was further amplified using goat IgG as blocking reagent and biotinylated anti-streptavidin antibody, followed by a second staining step with a streptavidin-phycoerythrin conjugate.
|
| Scan protocol |
Fluorescence was detected using the Affymetrix G3000 GeneArray Scanner and image analysis of each GeneChip was performed through the Affymetrix GeneChip Command Console version 2.0 (AGCC v2.0) software from Affymetrix, using the standard default settings.
|
| Description |
1359
IP DNA
|
| Data processing |
Affymetrix Human Tiling 1.0R Promoter Arrays were analyzed using Tiling Array Software (v 1.1.02, Affymetrix). The Raw data (intensity) were scaled to a target intensity of 100, normalized by quantile normalization, and log2 transformed.
|
| |
|
| Submission date |
Oct 01, 2020 |
| Last update date |
Jun 11, 2021 |
| Contact name |
Yun-Shien Lee |
| E-mail(s) |
[email protected]
|
| Organization name |
Ming Chuan Univ
|
| Department |
Dept. Biotechnology
|
| Lab |
GMRCL
|
| Street address |
TauYau
|
| City |
TauYau |
| State/province |
Taiwan |
| ZIP/Postal code |
115 |
| Country |
Taiwan |
| |
|
| Platform ID |
GPL5082 |
| Series (1) |
| GSE158894 |
To identify GCM1 novel target genes by chromatin immunoprecipitation-on-chip (ChIP-chip) analysis |
|
