RNAseq of Zea mays: earshoot 2-3 cm , br2tr1
Field-grown earshoots were harvested between 9-11am on sunny days of June 8th, 9th, and 13th of 2015.  Earshoot samples were rapidly harvested, measured, and immediately frozen in liquid nitrogen, pooled by date of harvest and size class and stored at -80C. Multiple earshoots were ground frozen in liquid nitrogen, followed by subsequent aliquoting of the frozen powder for multiple preparation replicates. Size A= pool of 15-20 earshoots of length 0.5-1 cm, size B= pool of 15-20 earshoots of length 1-2 cm, size C= pool of 8 earshoots of length 2-3 cm, and size D= pool of 8 earshoots of length 3-5 cm. Total RNA was extracted from ~0.1 g of frozen ground powder from the same aliquots used for MOA-seq, with replicates (10 total libraries: 2 per size class, with technical replicates for the two B-sized samples) using the Qiagen RNeasy Plant MiniKit (Qiagen 74904) following the manufacturers protocol. 400ng total RNA (determined by Qubit RNA HS reagents, Thermo) with a RIN >7 (Bioanalyzer RNA Nano, Agilent).   Libraries were prepared with the Biomek 400 Automated Workstation (Beckman Coulter), using the NEBNEXT Ultra II RNA Library Prep kit for Illumina (New England Biolabs) according to manufacturers instructions, with an RNA fragmentation time of 15 minutes, a 1/10th dilution of NEB adaptor and 11 cycles of PCR amplification with dual-indexing primers.  Amplified libraries were initially quantified by Qubit DNA HS reagents, checked for size and artifacts using Bioanalyzer DNA HS reagents and KAPA qPCR (KAPA Biosystems) was used to determine molar quantities of each library.  Individual libraries were diluted and pooled equimolar, and the pool was again checked by Bioanalyzer and KAPA qPCR before submission for paired-end 150bp sequencing at Novogene on an Illumina HiSeq 4000.