|
| Status |
Public on Sep 04, 2020 |
| Title |
ChIP-seq_MCF-7_Flag-AA-NRF-1_R2 |
| Sample type |
SRA |
| |
|
| Source name |
human breast cancer cell MCF-7
|
| Organism |
Homo sapiens |
| Characteristics |
morphology type: epithelial type of breast cancer cell
chip antibody/selection: Flag
|
| Treatment protocol |
MCF-7 and MCF-7/ADR cells were cultured in RPMI-1640 medium with 10% fetal calf serum. MCF-7/ADR cells were cultured in the presence of a low concentration of Dox (1 µg/ml) and passaged for 1 week in drug-free medium before the experiments. For labeling, media was aspirated and the cells were washed with PBS. DMSO stocks of GalNAz was added to achieve the final treatment conditions and incubated for 48 h, with vehicle-only controls always included. Full-length wild type (WT) human NRF1 and O-GlcNAc amino acid sites mutant (human NRF1, Ser447/Ser450 (conservative to mouse NRF1 Ser448/Ser451) → Ala) NRF1 were subcloned into pCMV-Puro64. Transfection of the MCF-7 and MCF-7/ADR cells was performed with Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. The stably transfected cells were then selected by the addition of puromycin to the medium. ~2x107 cells were used as a unit of cells for each experiment.
|
| Growth protocol |
RPMI-1640 medium with 10% fetal calf serum.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
MCF-7 and MCF-7/ADR cells were maintained in RPMI-1640 medium with 10% fetal calf serum. Approximately 2 x 10e7 cells were used for each COGC-seq, sWGA-seq or ChIP-seq assay. O-GlcNAz chromatin DNAs enriched by Streptavidin-magnetic beads were purified with the Qiagen PCR purification kit. For sWGA-seq, O-GlcNAc protein bound chromatin DNAs were enriched by sWGA-agarose beads. For NRF1, Flag-WT(wild type)-NRF1, or Flag-AA(Ser447/Ser450 → Ala)-NRF1 ChIP-seq, crosslinked chromatin complexes were immunoprecipitated with anti-NRF-1 antibody (protein A/G-magnetic beads) or anti-Flag-magnetic beads. For RNA-seq, total RNA was extracted from the MCF-7 and MCF-7/ADR cells using Trizol reagent (Invitrogen). RNA quality was checked by Agilent 2100 Bioanalyzer (Agilent Technologies, USA). RNA libraries were prepared for sequencing using standard BGI protocols.
DNA-end repair, 3'-dA overhang and ligation of methylated sequencing adaptor. PCR amplification and size selection (usually 100-300bp, including adaptor sequence. Qualified library for sequencing. RNA-seq libraries were constructed using SMARTer cDNA library construction kit (Clontech, Takara Bio, CA, USA).
ChIP-Seq/RNA-Seq
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
BGISEQ-500 |
| |
|
| Data processing |
base-calling: in-house BGISEQ-500 software
Bowtie2 (version: v2.2.5) was used to map clean reads to reference gene
peak-calling: MACS2 for ChIP-Seq; FPKM method was used in calculated RNA-seq expression level.
ChIPseeker for the annotation of peaks.
Genome_build: Genome_build: ChIP-Seq: hg19; RNA-Seq: hg19;
Supplementary_files_format_and_content: Supplementary_files_format_and_content: bigwig (bw): The signal intensity of binding.
|
| |
|
| Submission date |
Sep 03, 2020 |
| Last update date |
Sep 04, 2020 |
| Contact name |
bo yu liu |
| E-mail(s) |
[email protected]
|
| Phone |
15842607600
|
| Organization name |
Dalian University of Technology
|
| Street address |
Dagong Road 2
|
| City |
panjin |
| State/province |
liaoning |
| ZIP/Postal code |
124000 |
| Country |
China |
| |
|
| Platform ID |
GPL23227 |
| Series (1) |
| GSE141698 |
Genome-wide maps of O-GlcNAc proteins in human breast cancer cells by next generation sequencing |
|
| Relations |
| BioSample |
SAMN16054556 |
| SRA |
SRX9071262 |
