title: IR64 BSS&M LCM RNAseq
design: Rice cultivar IR64 (Oryza sativa ssp. indica) was germinated and grown in mixture of 1:1 topsoil and sand for about 2 weeks in a controlled environment growth room. The temperature was set at 28C day 25C night, with a photoperiod of 12 hr light and 12 hr dark, a relative humidity of 60% and a photon flux density of 300 mol m2 s1. The middle 1-cm sections of the fourth fully expanded leaves were sampled at 4 hours after dawn. Leaf tissue was fixed and embedded into Steedmans wax. Paradermal sections of 7 micron were prepared with microtome and mounted on PEN membrane slides. Prior to LCM, Steedmans wax was removed by incubating slides in 100% acetone for 1 min. LCM was performed on a an Arcturus Laser Capture Microdissection platform, isolated bundle sheath strands and mesophyll were collected using CapSure Macro Caps and RNA was extracted using PicoPure RNA Isolation Kit with on-column DNaseI treatment. 20-40 ng of BSS and M RNA was used for library preparation, rRNA was depleted using Ribozero kit, and then cDNA was amplified using Nugen RNA Ovation v2 RNAseq system according to user manual, after cDNA amplification, single strand cDNA was digested using S1 Nuclease (Promega), then 100 ng of amplified cDNA was sheared using Covaris E220 focused ultrasonicator, cDNA libraries were prepared using Illumina Truseqnano DNA library preparation kit.Paired-ended 2x 75-bp sequencing were performed using Nextseq 550 sequencer.