title: Hi-C-DpnII_control_replicate_1
design: An improved Hi-C protocol (Belton et al. 2012) was adapted for plant DNA and different restriction enzymes. Seedlings were fixed with 1% formaldehyde solution in MS buffer (10 mM potassium phosphate, pH 7.0; 50 mM NaCl; 0.1M sucrose) at room temperature for 30 min in vacuum. After fixation, the seedlings were incubated at room temperature for 5 min under vacuum in MC buffer with 0.15 M glycine. Around 2 g fixed tissue was homogenized with liquid nitrogen and resuspended in nuclei isolation buffer (Louwers et al. 2009), and filtered with 40 nm cell strainer. The procedures for enriching nuclei from flow-through, and denaturing nuclei were done according to a 3C protocol established for maize tissues (Louwers et al. 2009). The denatured chromatin was equally divided into five tubes and each was digested with 25 U of DpnII or HindIII overnight at 37?C. On the next day, digested DNA was blunt-ended by filling nucleotides by Klenow enzyme (Belton et al. 2012), during which biotin-14-dCTP (Invitrogen) was incorporated. In case of DpnII, an additional incubation at 65?C for 15 min was performed before the nucleotide fill-in reaction to inactivate this restriction enzyme. Next, the chromatin solution was diluted 10 times and blunt-end ligation was carried out in three tubes at 16?C overnight, with each tube having 100 Weiss units of T4 DNA ligase. On the next day, crosslinks were reversed in one tube by 65?C overnight incubation. The other two tubes were treated as controls; blunt-end ligation and reversion of crosslink were performed exactly the same but with reverse order. After ligation, DNA isolation and RNA digestion were performed according to (Belton et al. 2012).    Next, from Hi-C and control samples, 3 µg of DNA were incubated with T4 DNA polymerase to remove the biotin-14-dCTP from the end of un-ligated DNA molecules by following procedures described in (Belton et al. 2012). The protective nucleotides added were dTTP and dATP for DpnII, and dGTP and dATP for HindIII. DNA was then sheared by sonication with a CovarisTM S220; end repair and adenylation of DNA ends were performed according to (Lieberman-Aiden et al. 2009). Subsequently, the DNA was separated in 1.8% agarose gel and fragments from 300 to 600 bp were cut and recovered. Next, DNA was purified with Dynabeads® MyOne™ Streptavidin C1 beads (Invitrogen), and on-bead ligation with selected Illumina TruSeq Adaptor was done accordingly (Belton et al. 2012), except that 0.5 µl adaptor was used for each ligation. After washing, the beads were resuspended in 20 µl of 10 mM Tris-HCl buffer (pH 8.0).   Amplification of library molecules was performed according to the standard Illumina library preparation protocol. For a 50 µl PCR reaction, 5 µl of recovered Hi-C DNA bound to magnetic beads was used as template, and typically 10 cycles of amplification were used for Hi-C and 13 for control libraries. PCR products were purified and quantified with an Agilent 2100 Bioanalyzer, and sequenced on an Illumina Hiseq 2000 with 2x101 bp reads.