GSM2948297: cpm1mace; Solanum lycopersicum; RNA-Seq
Pollen was isolated from anthers by squeezing the stamens with a pipette tip 200 μl in the germination solution, followed by vortexing for 15 seconds. Pollen was passed through gauze cloth, washed twice with germination solution, and centrifuged at 4°C for 2 min at 100x g, followed by short spin to sediment the isolated pollen grains. The pellet was resuspended in 200 μl of germination solution, followed by centrifugation at 100 g for 2 minutes at 4°C, and followed by short spin. The supernatant was discarded and samples preserved in the liquid nitrogen. RNA was isolated from two biological replicates per developmental stage and condition using the Macherey-Nagel NucleoSpin miRNA isolation kit according to manufacturer's protocol. From the two resulting fractions the one comprising the large RNA (> 200 nt) was used for the next steps. The MACE was carried out according to the protocol described by Zawada et al. 2014 (PMID: 24184689): MACE libraries were prepared using the proprietary TrueQuant technology for elimination of PCR bias (Zawada et al. 2014). Poly-adenylated RNA was extracted with Dynabeads mRNA Purification kit (Life Technologies) from large RNA (>200 nt) and reverse transcribed with SuperScript Double-Stranded cDNA Synthesis Kit (Life Technologies) using biotinylated poly(dT) primers. Fragmentation of cDNA was conducted with Bioruptor (Diagenode) which yielded an average size of 250 bp. Biotinylated cDNA ends were captured by Dynabeads M-270 Streptavidin Beads (Life Technologies) and ligated with T4 DNA Ligase 1 (NEB) to modified adapters (TrueQuant, GenXPro). PCR amplification was performed with KAPA HiFi Hot-Start Polymerase (KAPA Biosystems), purified by Agencourt AMPure XP beads (Beckman Coulter) and sequenced with HiSeq2000 (Illumina) MACE (massive analysis of cDNA ends)
GEO Accession
GSM2948297