Brassinosteroids regulate the latex of rubber tree
1.1 RNA Quality AssessmentPurity, concentration and integrity of RNA sample were examined by NanoDrop, Qubit 2.0, Agilent 2100, etc. Only RNA with good quality could move on to following procedures.1.2 Library ConstructionQualified RNA were processed for library construction. The procedures are described as follow: (1) mRNA was isolated by Oligo(dT)-attached magnetic beads.(2) mRNA was then randomly fragmented in fragmentation buffer.(3) First-strand cDNA was synthesized with fragmented mRNA as template and random hexamers as primers, followed by second-strand synthesis with addition of PCR buffer, dNTPs, RNase H and DNA polymerase I. Purification of cDNA was processed with AMPure XP beads. (4) Double-strand cDNA was subjected to end repair. Adenosine was added to the end and ligated to adapters. AMPure XP beads were applied here to select fragments within size range of 300-400 bp. (5) cDNA library was obtained by certain rounds of PCR on cDNA fragments generated from step 4. 1.3 Library Quality ControlIn order to ensure the quality of library, Qubit 2.0 and Agilent 2100 were used to examine the concentration of cDNA and insert size. Q-PCR was processed to obtain a more accurate library concentration. Library with concentration larger than 2 nM is acceptable. 1.4 SequencingThe qualified library was pooled based on pre-designed target data volume and then sequenced on Illumina sequencing platform.2 Analysis FlowClean data with high quality was obtained by filtering Raw data, which removes adapter sequence and reads with low quality. These clean data were further mapped to pre-defined reference genome generating mapped data. Assessment on insert size and sequencing randomness were processed on mapped data as library quality control. Basic analysis on mapped data included gene expression quantification, alternative splicing analysis, novel genes prediction and genes structure optimization.