4.1. Hi-C Library Preparation and Sequencing

In situ Hi-C was performed as described previously [18] using frozen leaves from Medicago truncatula cv. R108. Briefly, frozen leaf tissue was crosslinked, ground and then lysed with nuclei permeabilized but still intact. DNA was then restricted with MboI restriction enzyme and the overhangs filled in incorporating a biotinylated base. Free ends were then ligated together in situ. Crosslinks were reversed, the DNA was sheared to 300–500 bp and then biotinylated ligation junctions were recovered with streptavidin beads.

Standard Illumina library construction protocol was used for DNA sequencing. Briefly, DNA was end-repaired using a combination of T4 DNA polymerase, Escherichia coli DNA Pol I large fragment (Klenow polymerase), and T4 polynucleotide kinase. The blunt, phosphorylated ends were treated with Klenow fragment (3′ to 5′ exo minus) and dATP to yield a protruding 3- ‘A’ base for ligation of Illumina’s adapters which have a single ‘T’ base overhang at the 3’ end. After adapter ligation, DNA was PCR amplified with Illumina primers for 14 cycles and library fragments of 400–600 bp (insert plus adaptor and PCR primer sequences) were purified using SPRI beads. The purified DNA was captured on an Illumina flow cell for cluster generation. Libraries were sequenced on the NextSeq500 following the manufacturer’s protocols. The same R108 lineage used for generating Tnt1 insertion lines was used for Hi-C. The resulting library was sequenced to yield approximately 48× coverage of the M. truncatula genome.