title: RNA-seq of ragweed
design: We stratified the seeds in moist silica sand and kept them in the dark at 4C for 10 weeks to break seed dormancy. After stratification, we planted the seeds in 50-cell trays in BM2 germination mix (Berger), and let them germinate in a growth chamber under a 14-hour day and a day/night temperature of 22/20C. We planted the seeds in a completely randomized design by including two to three seeds per cell. One week after planting, seedlings were randomly culled to one per cell. We grew the plants for 8 weeks, during which they were fertilized once, and watered as needed. After the 8 weeks, when each individual had between 8 and 12 leaves, we harvested the apical meristem and top four leaves from each plant. Tissue was collected on a single day over the course of one hour. We immediately flash froze the plant tissue in liquid nitrogen and then stored the samples at 80C. Total RNA was extracted from ground tissue (~50 mg) using a RNeasy Plant Mini Kit (Qiagen). The 74 total RNA samples were submitted to University of Minnesota Genomics Center for library preparation and sequencing, where a stranded TruSeq RNA library was created for each sample. The libraries were pooled and sequenced over three lanes on Illumina HiSeq 2500 in high output mode with 125-bp paired-end reads (~220 million reads per lane).