Transcriptome of Brassica juncea nectaries and leaves

Comparative RNA-Seq study of expression in B.juncea nectaries from different lines. Brassica juncea plants were grown on Sun Gro LC8 soil under a 16 hr day/8 hr night cycle, photosynthetic photon flux of 200 μmol m-2 s-1 at leaf level, and a temperature of 21°C. Three types of RNA samples were separately prepared from both the median and lateral nectaries of flowers, including: ‘pre-secretory’ (24 hours prior to anthesis/nectar secretion), ‘secretory’ (full anthesis, ~4-8 hours after dawn), and ‘post-secretory’ (12 hours after the ‘secretory’ stage). RNA was also isolated from leaf tissue collected at the same time as the nectary ‘secretory’ stage. All nectary tissues were separately dissected by hand from the flowers of primary inflorescences of ca. 40 day-old plants. Due to the small size of nectaries, dissections took place over several days from 4–8 hours after dawn (h.a.d.). Isolated nectaries were pooled in RNAlater™ solution (Ambion, Austin, TX) on ice, and stored at 4°C prior to RNA extraction. Up to four nectaries were collected per flower, with approximately 100 nectaries being processed as a single RNA sample. Each biological replicate was represented by nectaries pooled from different sets of plants. RNA was extracted from nectaries by mechanical disruption, with a microcentrifuge pestle, and using the RNAqueous®-Micro micro scale RNA isolation kit (Ambion, Austin, TX) with Plant RNA Isolation Aid (Ambion, Austin, TX). Agarose gel electrophoresis and UV spectrophotometry were used to assess RNA quality for all samples prior to submission to the University of Minnesota Genomics Center for barcoded library creation and Illumina HiSeq 2500 sequencing. Twenty-one TruSeq RNA v2 libraries were created (triplicate samples for both median and lateral nectaries at ‘pre-secretory’, ‘secretory’, and ‘post-secretory’ stages, as well as in triplicate for leaf) and sequenced via 50 bp, paired-end runs on the HiSeq 2500 using Rapid chemistry. All libraries were pooled and sequenced across two lanes to achieve the equivalent of 1 lane of output. This generated ≥162 M reads and the average quality scores were above Q30.