GA treatment early harvest rep1
Gene expression profiling was performed using a 3'-Tag-RNA-Seq protocol. Barcoded sequencing libraries were prepared using the QuantSeq FWD kit (Lexogen, Vienna, Austria) for multiplexed sequencing using both, the UDI-adapter and UMI Second-Strand Synthesis modules (Lexogen). The preparation followed the recommendations of the manufacturer with two modifications first being a reduction in fragmentation time to 10 s at 85C and the second was extending the reverse transcription step to 1 hour at 42C. The fragment size distribution of the libraries was confirmed via micro-capillary gel electrophoresis on a LabChip GX system (PerkinElmer, Waltham, MA). The libraries were quantified by fluorometry on a Qubit fluorometer (Life Technologies, Carlsbad, CA), and combined in equimolar ratios. The library pool was quantified via qPCR with a Kapa Library Quant kit (Kapa Biosystems/Roche, Basel, Switzerland) on a QuantStudio 5 system (Applied Biosystems, Foster City, CA). Up to forty-eight libraries were sequenced per lane on a HiSeq 4000 sequencer (Illumina, San Diego, CA) with single-end 100 bp reads.