title: Hi-C of Rosa Chinensis young leaves, lib 2
design: About 0.5 g of formaldehyde-fixed leaf tissues were used for the preparation of 2 independent in situ Hi-C libraries. Nuclei extraction, nuclei permeabilization, chromatin digestion, and proximity ligation treatments were performed essentially as previously described (Zhu, 2017). The extracted nuclei were resuspended in 150 µl 0.5% SDS, and split equally into three tubes. The nuclei were incubated at 62C for 5 min, after which 145 µl water and 25 µl 10% Triton X-100 were added, and incubated at 37C for 15 min. Next, the nuclei in each tube were digested by adding 25 µl 10x NEB buffer 3 (100 mM NaCl, 50 mM Tris-HCl, 10 mM MgCl2, 1 mM DTT, pH 7.9) and 50 U of DpnII restriction enzyme, and incubated at 37C overnight. On the next day, the nuclei were incubated at 62C for 20 min to inactivate the restriction enzyme. Next, the digested chromatin was blunt-ended by adding 1 µl of 10 mM dTTP, 1 µl of 10 mM dATP, 1 µl of 10 mM dGTP, 25 µl of 0.4 mM biotin-14-dCTP, 14 µl water and 4 µl (40 U) Klenow fragment, and incubated at 37ºC for 2 hr. 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 Weiss U T4 DNA ligase were added to start proximity ligation. The ligation reaction was placed at room temperature for 4 hr. After ligation, the nuclei were collected by spinning them down at 1000 rcf for 3 min, and then resuspended in 750 µl SDS buffer (50 mM Tris-HCl, 1% SDS, 10 mM EDTA, pH 8.0), and incubated with 200 µg proteinase K at 55C for 30 min. The formaldehyde crosslink was reversed by adding 30 μl 5M NaCl to the solution followed by overnight incubation at 65C. The recovery of Hi-C DNA and subsequent DNA manipulations were performed as described in Wang and al, 2015. The final libraries were sequenced on an Illumina NextSeq instrument with 2 x 75 bp reads.