RNA-seq of Brachypodium distachyon root tips growing in vitro, from 0 to 12h after root tip excision
Seeds were dehulled and surface sterilized in 6% (v/v) bleach containing 0.01% Triton X-100 (Sigma-Aldrich, Ref: X100-5ML) for 1 minute, then rinsed six times with sterile water. Sterilized seeds were sown on square 12  12 cm Petri dishes containing 1% plant agar (w/v) supplemented with half-strength Murashige and Skoog ( MS) basal medium and buffered with MES. No more than 15 seeds were placed in a single row approximately 3.5 cm from the top edge of each plate, with embryos facing upward and avoiding direct contact with the medium surface. Plates were placed vertically at an angle of ~20 in growth chambers under continuous light at 22C to promote root growth along the surface. For root tip excision experiments, 6-day-old seedlings had approximately 0.5 cm of the primary root tip removed using a sterile scalpel. After excision, plates were resealed and returned to the same growth conditions to allow for synchronized LR formation. For RNA sequencing, 5 mm of the root segment immediately proximal to the excision site was harvested at 0, 1, 2, 4, 8, and 12 hours after root tip excision and immediately flash-frozen in liquid nitrogen.