title: RNA-Seq of Arabidopsis pollen uncovers novel transcription and alternative splicing
design: For preparation of seedling samples, Arabidopsis (Arabidopsis thaliana) Col-0 seeds were sown on soil, kept at 4 deg  C for 3 d to encourage uniform generation, and then transferred to a temperature-controlled growth room set to  22 deg C. Plants were grown in a 16-h/8-h light/dark cycle under 100 to 120 PAR.   After 21 d of growth, the aerial portions of  the seedlings were collected twice per day for 5 d and pooled for RNA extraction.  The experiment was then repeated following the same collection  scheme, thus providing two distinct biological replicates. Mature, dry pollen  was harvested from Col-0 plants using a vacuum collection device as described  (Johnson-Brousseau and McCormick, 2004). For preparation of RNA, seedlings or pollen were ground into a fine powder with a mortar and  pestle, and RNA was isolated via TRI-reagent extraction with cleanup on  Qiagen Plant RNeasy (catalog no. 74104) columns. All RNAs were treated with  DNaseI using the Plant RNeasy Kit. A starting amount of 15 micrograms of total RNA  from each sample (seedling and pollen) was used in the library preparation,  using the Illumina mRNA-seq Sample Preparation Kit (catalog no. RS-930-  1001, part no. 1004898). mRNA was isolated and purified via Sera-Mag  Magnetic Oligo(dT) Beads, washed, and then fragmented using divalent cations  under elevated temperature. First- and second-strand cDNAs were synthesized,  and an end-repair step was performed to convert overhangs into  blunt ends. Next, the 3-prime ends were adenylated for ligation of the Illumina  adapters. cDNA templates were then purified by gel isolation for a size selection  of approximately 250 bp and amplified via PCR so that an adequate  amount of sample was available for sequencing. Products were isolated with the Qiagen QIAquick PCR Purification Kit (catalog no. 28104) and added to the flow cell for sequencing.