Transcriptome sequencing, annotation, and analysis
Tomato leaflet samples were collected from JZ8, 35S:rSlAGO1 , and35S:SlmiR-168a at the same stage and position on the plants, and
an Illumina Miseq library was constructed (Illumina), according to the
manufacturer’s instructions. Magnetic beads with poly T oligos attached
were used to purify the mRNA from the total RNA. Fragmentation buffer
was added to cleave the mRNA into short fragments, and the fragments
were used to synthesise first-strand cDNA using random hexamer primers.
The cDNA was transformed into double-stranded cDNA with RHase H and DNA
polymerase I, and a paired end library was constructed from the
synthesised cDNA with a Genomic Sample Prep Kit (Illumina). Fragments of
desirable lengths were purified with a QIAquick PCR Extraction Kit
(Qiagen, Valencia, CA, USA), end repaired, and linked with sequencing
adapters (Margulies et al. 2005). AMPureXP beads were used to remove the
unsuitable fragments, and the sequencing library was the constructed by
PCR amplification. Libraries were checked with PicoGreen staining and
fluorospectrophotometry, quantified with an Agilent 2100 instrument, and
mixed in equal volumes to a normalised concentration of 10 nM. The
sequencing library was then sequenced using the Illumina Miseq platform
(LC-BIO Technology Co., Ltd.).