GSM6862089: Hi-C_8h_rep1; Oryza sativa Indica Group; Hi-C
In total, 0.5 g of homogenized tissue powder produced via grinding fixed tissues with liquid nitrogen was used for one round of library prep. The extracted nuclei were resuspended with 150 µl 0.5% SDS and split equally into three tubes. The nuclei were incubated at 62 °C for 5 min, after which the SDS molecules were quenched by adding 145 µl water and 25µl 10% Triton X-100, and incubated at 37 °C for 15 min. The DNA in the nuclei in each tube was digested by adding 25µl 10x NEB buffer 3 (100 mM NaCl, 50 mM Tris-HCl, 10 mM MgCl2, 1 mM DTT, pH 7.9) and 50 U of MboI, and incubated at 37 °C overnight. On the next day, the MboI enzyme was inactivated at 62 °C for 20 min. Next, the cohesive ends were filled in by adding 1 µl of 10 mM dTTP, 1 µl of 10 mM dATP, 1 µl of 10 mM dGTP, 25µl of 0.4 mM biotin-14-dCTP, 14µl water and 4µl (40 U) Klenow, and incubated at 37 °C for 2 h. Subsequently, 663 µl water, 120 µl 10x blunt-end ligation buffer (300 mM Tris-HCl, 100 mM MgCl2, 100 mM DTT, 1 mM ATP, pH 7.8), 100 µl 10% Triton X-100 and 20 U T4 DNA ligase were added to start proximity ligation. The ligation reaction was placed at room temperature for 4 h. Next, the reaction mixture was centrifuged at 1000 g for 3 min, and the nuclei pellet was resuspended with 750 µl SDS buffer (50 mM Tris-HCl, 1% SDS, 10 mM EDTA, pH 8.0) and incubated with 200 µg proteinase K at 55 °C for 30 min. The formaldehyde cross-link was reversed by adding 30 μl 5 M NaCl to the solution followed by incubation at 65 °C overnight. Amplification of library molecules was performed according to the standard BGI library preparation protocol. The final library was sequenced on a BGI MGISEQ-2000 instrument with 2 ×150 base pair (bp) reads.