title: Nodal Root Bud Development - Transcriptome Analysis
design: Nodal Root Bud Development - Transcriptome Analysis
library_construction_protocol: At 120 days after emergence (DAE), five TX08001 field-grown plants were harvested for morphological analysis and RNA-seq. Leaf blades were removed for transport, and in the laboratory, leaf sheaths were dissected using a scalpel to expose the stem tissue. The relative position, number, and size of nodal root buds (NRBs) on each stem node were measured. NRB tissue samples were collected from stem nodes of phytomers 7-21 by excising a 3-4 mm deep wedge of pulvinus rind tissue containing the NRBs. Three replicate plants were sampled, and mid-internode stem rind samples from phytomers 6, 8, 9, 20, and 21, where NRBs do not form, were collected as controls. All tissue samples were flash-frozen in liquid nitrogen and stored in Whirl-Pak速 bags for further analysis. In addition, plants containing 7 phytomers with fully elongated internodes were harvested at 40 DAE to investigate the number of NRB rings on the nodes of plants containing less than 17 developed phytomers. The bioenergy sorghum hybrid TX08001 was planted at the Texas A&M University Farm in Burleson County, TX on 4/29/2021; plant emergence occurred 6-days after planting on 5/5/2021. Plot size was thirty-two 30 m long rows at a row spacing of 76 cm. At the time of planting, a solution of liquid ammonium polyphosphate (11-37-0), UAN 32%, and zinc sulfate was applied at a depth of 5 cm to the side of the seed for a total fertilizer yield of 45-63-0 + 5 Zn kg/ha. The soil consisted of Weswood Silt Loam (Staff, 2022). The plot was sown with enough seeds to allow thinning to 15 cm plant spacing within rows at 21 days after emergence (DAE). Seeds, before planting, were treated with Concept III速, a herbicide protectant, Nugro速 a systemic insecticide and Apron X速, a systemic fungicide. Tissues were ground to a fine powder with a heat sterilized mortar and pestle filled with liquid nitrogen then transferred into liquid- nitrogen chilled sterile 1.5 mL c centrifuge tubes. RNA was extracted using the Zymo RNA Mini- Prep kit. Purity and concentration of the RNA was analyzed using a Thermo ScientificTM NanoDrop One Microvolume UV-Vis Spectrophotometer before being sent for fragmentation analysis on an Agilent 5300 Fragment Analyzer using software version 3.1.0.12. Plate-based RNA sample prep was performed on the PerkinElmer Sciclone NGS robotic liquid handling system using Illumina's TruSeq Stranded mRNA HT sample prep kit utilizing poly-A selection of mRNA following the protocol outlined by Illumina in their user guide: https://support.illumina.com/sequencing/sequencing_kits/truseq-stranded-mrna.html, and with the following conditions: total RNA starting material was 1000 ng per sample and 8 cycles of PCR was used for library amplification. The prepared libraries were quantified using KAPA Biosystems' next-generation sequencing library qPCR kit and run on a Roche LightCycler 480 real-time PCR instrument. Sequencing of the flowcell was performed on the Illumina NovaSeq sequencer using NovaSeq XP V1.5 reagent kits, S4 flowcell, following a 2x151 indexed run recipe.