title: NextSeq 500 sequencing; RNAseq of wt and receptor mutant lines in response to 7 elicitor treatments over a 6-point time course
design: RNAseq of wt and receptor mutant lines in response to 7 elicitor treatments over a 6-point time course
library_construction_protocol: Seedlings for collection were in 24-well plates. Each well contained two seedlings; two wells and four seedlings were combined for one sample. Samples were quickly blotted dry and flash-frozen in liquid nitrogen. Arabidopsis seeds were surface-sterilized, stratified 3-5 days, grown 4 days in 22 °C, 16 h/8 h light/dark on full strength MS agar plates, 1% sucrose. 4-day-old seedlings were transferred to full strength liquid MS media, 1% sucrose, and grown for 9 more days. When plants were 13 days old, media was exchanged, and after overnight incubation elicitor was added to individual wells. Elicitor treatment was added as 400 uL 2.5x concentration in liquid MS medium to 600uL liquid MS medium, resulting in 1 mL 1x concentration. mRNA was extracted from plant lysate through pull-down of poly-A tail, as described in  \"BrAD-seq: Breath Adapter Directional sequencing: a streamlined, ultra-simple and fast library preparation protocol for strand specific mRNA library construction\" Townsley et al Frontiers in Plant Science 2015. dscDNA was made as in \"A high-throughput method for Illumina RNA-Seq library preparation\" Kumar et al Frontiers in Plant Science 2012 Libraries were simultaneously fragmented and tagged using a hyperactive Tn-5 as in \"Tn5 transposase and tagmentation procedures for massively scaled sequencing projects\" Picelli et al 2014 Genome Research and \"High‐resolution Expression Profiling of Selected Gene Sets during Plant Immune Activation\" Ding et al Plant Biotechnology Journal 2020