Time-lapse RNA-seq analysis to study transcriptional regulation by early signals in De Novo Root Regeneration in Arabidopsis

Detached Arabidopsis leaves can regenerate adventitious roots, providing a platform to study de novo root regeneration (DNRR). We performed time-lapse RNA-seq within 12 h to reveal transcriptional changes in response to early signals in DNRR. Arabidopsis seeds (Col-0 and the coi1-2 mutant) were germinated and grown on 1/2 MS medium with 1% sucrose at 22°C under a 16-h light/8-h dark photoperiod for 12 d. The first pair of rosette leaves from 12-d-old seedlings were cut at the junction of the blade and petiole, and the detached blades were cultured on sucrose-free B5 medium at 22°C under 24-h light conditions. The entire tissues of leaf explants at time 0 (t0), 10 min, 30 min, 1 h, 2 h, 4 h, 8 h and 12 h after detachment were harvested and frozen in liquid nitrogen for RNA extration. Library construction and deep sequencing were carried out using the Illumina HiSeq 3000 platform following the manufacturer's instructions by Genergy Biotechnology (Shanghai, China).