|
| Status |
Public on Apr 29, 2021 |
| Title |
HEK293 WT NUP98-HOXA9 (HA) Input (matched for CTCF IP) |
| Sample type |
SRA |
| |
|
| Source name |
HEK 293 cells
|
| Organism |
Homo sapiens |
| Characteristics |
antibody used: n/a (input)
treatment condition: 3XHA-3XFLAG-tagged, IDR-WT NUP98-HOXA9
|
| Treatment protocol |
For ChIP-seq experiments, HEK293 cells with stable expression of IDR-WT or IDR-mutated NUP98-HOXA9 and FUS-HOXA9 (GFP-tagged or 3XHA-3XFLAG-tagged) were grown in DMEM medium and fixed with 1% of formaldehyde in PBS for 10 min at room temperature. For 1,6-Hexanediol treatment, growth medium was removed and cells then incubated with 10% of 1,6-hexanediol in PBS for 1 min. Then, formaldehyde was added directly to make up the final concentration formaldehyde to be 1%, followed by incubation at room temperature for 10 min.
|
| Growth protocol |
HEK 293 cells were grown in DMEM base medium plus 10% FBS and 1% antibiotics. The fusion-transformed mouse HSPCs (leukemia-causing cells) were grown in Opti-MEM base medium plus 10% FBS, 2% of supernatant containing home-made murine stem cell factor (from mSCF-producing CHO cells), 1% of supernatant containing murine FLT3 ligand (from FLT3L-producing cells), 1% antibiotics and 50uM of beta-mercaptoethanol.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Fixed cells were sonicated in a Bioruptor sonication device for 45 cycles at a high-energy setting to enrich the sonicated DNA to be ~300bp as described in our method. The sonicated samples were incubated with the indicated antibodies pre-bound to the protein-G/A beads for overnight. Across 293 cell samples with GFP-tagged chimera, the cleared cell lysates from sonicated nuclei were added with the same fraction of the sonicated and cleared chromatin of Drosophila S2 cells (see our Method), and then protein-DNA complexes were isolated with antibodies against GFP and those against Drosophila-specific histone H2Av (Active Motif cat# 39715). Beads were then washed with various washing buffers (please see our method). Bead-bound DNA was eluted and subject to reverse crosslinking and sequential treatments of RNase and protease K. Finally, ChIP DNA was purified with a PCR cleanup kit (Qiagen) and used for library construction.
Multiplexed ChIP-seq sequencing libraries were prepared using the NEBNext Ultra II kit (NEB, #E7645L) following the manufacture’s instruction. ChIP-seq libraries were pooled together and sequenced on a Nextseq 550 system using a Nextseq 550 High Output Kit v2.5 or sent to UNC High-Throughput Sequencing Facility (HTSF) for sequencing by using an Illuminia Hiseq 2500/4000 system.
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| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
NextSeq 550 |
| |
|
| Data processing |
All reads were mapped to the human genome (hg19), drosophila (dm3) or mouse genome (mm10) using the BWA alignment software. Unique reads mapped to a single best-matching location with no more than two mismatches were kept, which were then subject to removal of reads generated by PCR- caused duplicates using the Picard and Samtools. We filter out reads with a mapping quality score of < 20 for downstream analysis.
Profiles of ChIP-seq read densities were displayed in the Integrative Genomics Viewer (IGV, Broad Institute).
The MACS2 software was used for peak identification with data from input as controls and default parameters.
Genome_build: GRCh37/hg19 or GRCm38/mm10
|
| |
|
| Submission date |
Apr 28, 2021 |
| Last update date |
Apr 30, 2021 |
| Contact name |
Gang Greg Wang |
| E-mail(s) |
[email protected]
|
| Phone |
919-9665952
|
| Organization name |
UNC Lineberger Comprehensive Cancer Center
|
| Department |
Dept of Biochemistry and Biophysics
|
| Lab |
gregwanglab
|
| Street address |
450 West Drive
|
| City |
Chapel Hill |
| State/province |
NC |
| ZIP/Postal code |
27599-7295 |
| Country |
USA |
| |
|
| Platform ID |
GPL21697 |
| Series (2) |
| GSE144076 |
Phase separation drives aberrant chromatin looping and cancer development [ChIP-Seq] |
| GSE144643 |
A phase separation mechanism underlies development of cancer and aberrant organization of three-dimensional chromatin structure |
|
| Relations |
| BioSample |
SAMN18914779 |
| SRA |
SRX10704237 |
