title: GSM8721340: HAP1,Micro-C rep1; Homo sapiens; MNase-Seq
library_construction_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.
identifiers.namespace: GEO
identifiers.id: GSM8721340_r1