title: NextSeq 500 paired end sequencing; EVOREPRO: RNA- seq of Arabidopsis thaliana pollen developmental stages for Ler and Col ecotypes
library_construction_protocol: Mature pollen grains (MPG) were harvested from open flowers of 5 to 6-week old plants with a modified vacuum cleaner using 100, 53 and 5 µm mesh (Johnson-Brousseau and McCormick, 2004) before the isolation of pollen developmental stages. Microspores and developing pollen grains were released from anthers of closed flower buds and purified by Percoll density gradient centrifugation as described (Dupláková et al., 2016; Honys and Twell, 2004). Populations of spores at five stages of development were isolated: uninucleate microspores (UNM), bicellular pollen (BCP), late bicellular pollen (LBC), tricellular pollen (TCP) and mature pollen (MPG).  For semi in vivo pollen tube growth, a transgenic marker line harboring MGH3p::MGH3-eGFP and ACT11p::H2B-mRFP (Borges et al. 2012) was used to pollinate WT emasculated pistils. After 2 hours, the pollinated pistil was excised and placed on double sided tape. The excised pistil was then cut at the junction of style and ovary and placed gently on solidified agarose pollen germination medium (Boavida and McCormick, 2007). The pistil was incubated for an additional 4 hours for the pollen tubes to emerge from the cut end of the style. The pollen tubes were harvested using 25G needle and immediately frozen in liquid nitrogen and subsequently used for the RNA-seq library preparation as described in Misra et al., 2019. Arabidopsis thaliana accession Columbia-0 (Col-0) plants were grown in controlled-environment cabinets at 21°C under illumination of 150 μmol/m2/sec with a 16-h photoperiod.  Total RNA was isolated from each sample using the RNeasy Plant Kit (Qiagen) according to the manufacturer's instructions. RNA was DNAse-treated (DNA-freeTM Kit Ambion, Life Technologies) according to the manufacturer's protocol. RNA yield and purity were determined spectrophotometrically and using an Agilent 2100 Bioanalyzer.  cDNA was prepared using a slightly modified SmartSeq2 protocol in which cDNA is synthesized from poly(A)+ RNA with an oligo(dT)-tailed primer (Picelli et al., 2013; 2014). The final libraries were prepared using a low-input Nextera protocol (Baym et al., 2015).