Dynabeads

In situ Hi-C was carried out as described^{5 (link)}. Cells were crosslinked with 1% formaldehyde then lysed to collect nuclei. Pelleted nuclei were digested with DpnII restriction enzyme (NEB, R0147). The restriction fragment overhangs were filled and marked with biotin-labelled dATP (Thermo Fisher, 19524016) and dCTP, dTTP, and dGTP before ligation. DNA was reverse crosslinked, purified, and fragmented by sonication on a Covaris sonicator. Biotin-labelled DNA was pulled-down on Streptavidin Dynabeads (NEB, S1420S). After DNA repair and 3′ A addition, SHORT Y-Adaptor (Supplementary Table 1) was added. Diluted DNA on Dynabeads was used for PCR amplification (4, 8, 12,16, and 20 cycles) to produce similar amounts of DNA for sequencing on the Illumina HiSeq X10 platform (paired end 2 × 150 bp reads).

We performed TSS-seq on 14-day-old seedlings grown on MS plates following a previously published protocol^{44 (link),65 (link)}. We applied heat stress by treating plants for 2 h at 37 °C. 5 µg of total RNA were treated with DNase and CIP (NEB) to remove DNA and all non-capped RNA. Then 5’ caps of capped RNA were removed with Cap-Clip (CellScript). We then ligated single-stranded rP5_RND adapters to 5’-ends with T4 RNA ligase 1 (NEB). Ligated RNAs were enriched and captured by oligo(dT) Dynabeads (Thermo Fisher Scientific). We fragmented Enriched samples for 5 min at 80 °C and generated the first-strand cDNA with SuperScript III (Invitrogen) and random primers. Second-strand cDNA was synthesized with Phusion High-Fidelity DNA Polymerase (NEB) and the BioNotI-P5-PET oligo and captured by Dynabeads for end repairing with End Repair Enzyme Mix (NEB) and ligation with barcoded Illumina compatible adapter using T4 DNA Ligase (NEB). We amplified TSS-seq sequencing libraries and size selected for single-end sequencing with NovaSeq 6000 platform (Illumina).