rice leaf gradient lcm-seq
For laser capture microdissection, leaf blade were separated from midrib and then embedded in Steedmans wax as previously describe (Hua and Hibberd, 2019). Microtome was used to create 7 m paradermal sections which were then mounted on polyethylene naphthalate (PEN) membrane slides using DEPC treated water (Hua and Hibberd, 2019). Steedman's wax was removed by exposing the slides to 100% acetone for 1 minute prior to LCM, to distinguish bundle sheath in younger leaf tissue (stage 2, 3, and 4), slides were incubated in 1% iodine in acetone for 1 min. Using the Arcturus LCM platform, isolated cells were collected on CapSure Macro Caps, and RNA was extracted using the PicoPure RNA Isolation Kit with on-column DNase I treatment. RNA quality and concentration were analysed using an Agilent Bioanalyser RNA 6000 Pico assay. RNA from at least three different leaves were combined into one biological replicate, 1.4-3ng of bundle sheath strands or mesophyll RNA with three to four biological replicates for each stage were reverse transcribed and amplified using the SMART-Seq v4 Ultra Low Input RNA Kit (Clontech) according to the manufacturers instructions, 11 PCR cycles were used for all samples. 1 ng of amplified cDNA was used as input for preparing library using the Nextera XT DNA Library Preparation Kit (Illumina). Libraries were sequenced using Illumnias NovaSeq 6000 sequencing platform to generate 31-50 million paired-ended 150-bp reads for each sample.