title: GSM3307879: Immature pollen grains-ATT conditions (IPGs_ATT); Solanum lycopersicum; RNA-Seq
library_construction_protocol: For transcriptome analysis, pollen grains were derived from plants that were maintained under control conditions and from plants that were exposed to the above-detailed ATT conditions. For each pollen sample, anthers collected from 90 flowers, at developmental stages of three, two, one and zero days before anthesis were dissected and pollen grains were obtained by slicing the anthers transversely and vortexing them in cold, sucrose-free germination solution. The solution was then filtered and pollen grains separated. The released pollen grains were immediately frozen in liquid nitrogen and kept frozen at -80 oC until use. Pollen grains were ground to a fine powder using liquid nitrogen and sea sand (Merck, Darmstadt, Germany) and total RNA was extracted using the Tri-reagent (Sigma Aldrich, Israel). Before further analyses, RNA derived from three stages of pollen maturation (three, two and one days before flower opening, corresponding to post-meiotic immature pollen grains; IPGs) was combined, using equal amounts, into one pool, generating a pooled sample of IPGs RNA, in addition to RNA isolated from mature pollen grains (zero days before flower opening; MPGs), that constituted a separate sample. For preparation of MACE libraries, poly-adenylated mRNA was isolated from 1 μg of the large fraction of total RNA using Dynabeads® mRNA Purification Kit (Life Technologies GmbH) and cDNA was produced by first and second strand synthesis using the SuperScript® III First-Strand Synthesis System (Life Technologies GmbH). , but with modified, barcoded poly-T adapters that are suitable to bind on the Illumina Hiseq2000 flow cell and that are biotinylated at the very 5′ end. After cDNA production, the biotinylated cDNA was random-fragmented to reach an average size of 250 bps, the 3′ends were captured by streptavidin beads and GenXPro's TrueQuant adapters were ligated. Ten barcoded samples were sequenced simultaneously in one lane of an Illumina Hiseq2000 with 1 × 94 bps.
attributes.tag: GEO Accession
attributes.value: GSM3307879