Paired end adapter ligation

For transcriptome sequencing, total RNA isolated from different tissues (root, stem, leaf and flower) of V. galamensis was equally mixed together to form a mixed sample (T01), which was used to construct cDNA library. Importantly, seed samples of three representative stages of V. galamensis seed development at 17 DAP (T02), 38 DAP (T03), and 45 DAP (T04) were harvested, respectively, for cDNA library construction. Total RNA from each sample was isolated using Trizol Reagent (Shenggong, China) following the manufacturer’s protocol. After checking the quality and quantity with NanoDrop spectrophotometer (NanoDrop Technologies, USA) and Agilent 2100 Bioanalyzer (Agilent Technologies, USA), mRNA was enriched using oligo (dT) beads and then broken into short pieces by random shearing. We further used these fragments as templates for first and second strand cDNA synthesis. Obtained cDNA fragments were then processed with end repair, Illumina’s paired-end adapter ligation, size range selection, purification and PCR amplification. Finally, 12 paired-end cDNA libraries with three biological replicates in each indicated sample were constructed and then sequenced on flow cell using Illumina HiSeq™ 2500 system in Biomarker Technology Corporation (Beijing, China).

Fresh leaves were cut into 2-cm pieces and fixed with 2% formaldehyde. Hi-C library construction was performed as previously described^{23 (link)}. In brief, genomic DNA was extracted, and the fixed chromatin was digested by DpnII, followed by a fill-in reaction with biotinylated nucleotides and proximity ligation. The DNA was purified and then sheared into ~ 350-bp fragments using a Covaris S220 device. The DNA fragments were subjected to blunt-end repair, A-tailing, Illumina paired-end adapter ligation and PCR amplification. The Hi-C library was sequenced on an Illumina NovaSeq PE150 instrument.