 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on Jan 10, 2025 |
| Title |
HAP1,Micro-C rep6 |
| Sample type |
SRA |
| |
|
| Source name |
cell line
|
| Organism |
Homo sapiens |
| Characteristics |
tissue: cell line
cell line: HAP1
enzyme: Mnase
|
| Treatment protocol |
Diploid HAP1 cells were isolated from Hoechst (MCE)-stained HAP1 cells by flow sorting.
|
| Growth protocol |
HAP1 (Horizon Discovery) cells were cultured in IMDM (ThermoFisher) supplemented with 10% fetal bovine serum in 5% CO2 at 37°C.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
The Micro-C assay was carried out in diploid HAP1 cells, using a published protocol.
Micro-C library preparation was prepared as previously described(Hsieh et al., 2020; Hsieh et al., 2016). Briefly, HAP1 cells (~1.5 × 10⁷) were fixed in 1% formaldehyde (Sigma-Aldrich, F8775) in PBS at room temperature for 10 min. Crosslinking was quenched with 1 M Tris-HCl (pH 7.5) to a final concentration of 375 mM for 5 min at room temperature. Cells were spun down and washed twice with cold PBS. Cell pellets were crosslinked again with freshly prepared 3 mM DSG (ThermoFisher, 20593) crosslinking solution for 45 min at RT. Crosslinking termination and washing is the same as in the previous step. Crosslinked cell pellets were resuspended in MB#1 (50 mM NaCl, 10 mM Tris-HCl pH = 7.5, 5 mM MgCl2, 1mM CaCl2, 0.2% NP-40, 1x Roche cOmplete EDTA-free (Roche diagnostics, 04693132001)) at a concentration of 1x106 cells/100 µL and were incubated for 20 min on ice. Then wash nuclei pellet in MB#1 at a concentration of 1x106 cells/100 μL. Centrifuge at ~2,000 x g for 5 min at 4°C. Added the appropriate amount of MNase (as determined by an MNase titration experiment on the same sample batch) to digest chromatin to an 80-90% monomer / 20-10% dimer ratio. The MNase reaction was inactivated by adding 4 mM EGTA and incubated for 10 min at 65°C. Digested chromatin was washed twice with 1 mL of cold MB2 (50 mM NaCl, 10 mM Tris-HCl pH 7.5, 10 mM MgCl2). Next, the pellet was resuspended in the end-repair buffer (50 mM NaCl, 10 mM Tris-HCl pH 7.5, 10 mM MgCl2, 100 µg/mL BSA, 2 mM ATP, 5 mM DTT) and 25 units of T4 Polynucleotide Kinase (NEB, M0201) for 15 min at 37°C while shaking in a thermomixer at 1000 rpm. Then, we added 25 units of DNA Polymerase I, Large (Klenow) Fragment (NEB, M0210) directly to the reaction and incubated the tube for an additional 15 min at 37°C while shaking in a thermomixer at 1000 rpm. Finally, in order to repair the cut end of the enzyme to form a blunt and ligatable ends, we supplemented 66 µM of dNTPs (dTTP, dGTP (NEB, N0446), biotin-dATP (Jena Bioscience, NU-835-BIO14), biotin-dCTP (Jena Bioscience, NU-809-BIOX), 100 µg/mL BSA and 1X T4 DNA ligase reaction buffer (NEB, B0202) directly into the reaction and incubated for 45 min at 25°C while shaking in a thermomixer at 1000 rpm. The end-repair reaction was then inactivated with 30 mM EDTA for 20 min at 65°C without shaking. Next, chromatin was pelleted washed once with cold MB3 (50 mM Tris-HCl pH 7.5, 10 mM MgCl2). End-repaired nucleosomes were then subjected to proximity ligation with ~5000 U of T4 DNA ligase (NEB, M0202) in 1X T4 DNA ligase reaction buffer (NEB, B0202) for at least 2 hours at RT with slow rotation using a gentle nutator. The unligated ends of biotin-dNTPs were removed with ~500 units of Exonuclease III (NEB, M0206) in 1X NEBuffer 1 (NEB, B7001) for 15 min at 37°C while shaking in a thermomixer at 1000 rpm. The samples were digested by protease K and DNA was extracted with phenol chloroform.250-350bp DNA was purified by 2% agarose gel. Using Dynabeads MyOne Streptavidin C1 beads (ThermoFisher, 65001) to capture biotinylated DNA fragments. The VAHTS Universal DNA Library Prep Kit for Illumina V3 (Vazyme, ND607-01) was used to construct the library. The Micro-seq library were sequenced on the Illumina NovaSeq 6000 platform, producing pair-end reads of 150 bp.
|
| |
|
| Library strategy |
MNase-Seq |
| Library source |
genomic |
| Library selection |
MNase |
| Instrument model |
Illumina NovaSeq 6000 |
| |
|
| Description |
Library name: MicroC-HAP1 rep 6
|
| Data processing |
The micro-C data was analyzed according to the procedures outlined in the documentation at https://micro-c.readthedocs.io/en/latest/. Initially, raw sequencing reads underwent quality checks and filtering to eliminate low-quality sequences. The remaining reads were then aligned to the hg19 reference genome using BWA-MEM. After alignment, the data was processed to identify contact interactions by pairing aligned reads, sorting, and filtering for low-quality events, PCR duplicates, and invalid pairs using Pairtools (https://pairtools.readthedocs.io/en/latest). These filtered reads were then assigned to interaction pairs. Finally, chromatin loops were identified using the HiCCUPS loop-calling program at 10 kb and 5 kb resolutions, utilizing hic files generated from unique valid read pairs.
Assembly: hg19
Supplementary files format and content: bedpe
|
| |
|
| Submission date |
Jan 09, 2025 |
| Last update date |
Jan 10, 2025 |
| Contact name |
Ke Zhao |
| E-mail(s) |
zk8584918@gmail.com
|
| Organization name |
Tianjin Medical University
|
| Street address |
Qixiangtai Road, No.22
|
| City |
Tianjin |
| ZIP/Postal code |
05000 |
| Country |
China |
| |
|
| Platform ID |
GPL24676 |
| Series (2) |
| GSE286224 |
Endogenous fine-mapping of functional regulatory elements in complex genetic loci in HAP1 [microC] |
| GSE286226 |
Endogenous fine-mapping of functional regulatory elements in complex genetic loci in HAP1 |
|
| Relations |
| BioSample |
SAMN46179772 |
| Supplementary data files not provided |
| Raw data are available in SRA |
|
|
|
|
 |
