title: Illumina HiSeq 2500 paired end sequencing; RNA-seq of Solanum lycopersicum pollen tube at different germination temperatures. EVOREPRO dataset
library_construction_protocol: Dry pollen grains were collected from stage 15 flowers (Brukhin et al., 2003) into 500µl eppendorf tubes. Pollen tubes were grown in 300µl of pollen growth medium in a 750µl eppendorf tube that was incubated in a 28°C water bath.  Pollen tubes were grown at a density of ~1000 pollen grains/µl. The pollen germination medium (Covey et al., 2010) comprised 24% (w/v) polyethylene glycol (PEG) 4000, 0.01% (w/v) boric acid, 2% (w/v) Suc, 20 mm MES buffer, pH 6.0, 3 mm Ca(NO3)2·4H2O, 0.02% (w/v) MgSO4·7H2O, and 1 mm KNO3. Pollen tubes were grown for 1.5 hours, 3 hours, or 9 hours before they were collected by centrifugation (1000 x g) for 1 minute. Pollen germination medium was carefully removed by pipetting to avoid disrupting the loose pollen tube pellet. Independent pollen collections were made for each of three biological replicates at each time point.  Solanum lycopersicum (tomato accession Nagcarlang, LA2661) seeds were obtained from the Tomato Genetics Resource Center (TGRC, https://tgrc.ucdavis.edu/) and grown in the Brown University Greenhouse (Providence, RI, USA).  Eppendorf tubes containing pollen tubes were immediately flash frozen in liquid N2, then stored at -80°C, or put directly on a dry-ice cooled metal block for cell disruption by grinding with a frozen plastic pestle (Kontes). Total RNA was extracted using the RNeasy Plant Kit (Qiagen).