F_K326_S3_Leaf_1
Samples for RNA sequencing were collected in 2022. Three replicates of each plant were sent to BGI for RNA-seq on the BGISEQ-500 platform and bisulfate-seq. mRNA was purified using poly-T oligo-attached magnetic beads, fragmented at high temperature with divalent cations, and converted to first-strand cDNA with reverse transcriptase and random primers. Second-strand cDNA was synthesized using DNA Polymerase I and RNase H, followed by adapter ligation after adding an 'A' base. Products were purified and enriched via PCR amplification.PCR yield was quantified by Qubit, and samples were pooled to form single-stranded DNA circles (ssDNA circles) for the final library. ssDNA circles underwent rolling circle replication (RCR) to generate DNA nanoballs (DNBs), which enhanced fluorescent signals during sequencing(Zhai et al. 2020). DNBs were loaded onto patterned nanoarrays, and 100 bp (or 150 bp) paired-end reads were performed on the BGISEQ-500 platform (Fehlmann et al. 2081) using the Combinational Probe-Anchor Synthesis Sequencing Method for subsequent data analysis .
SUB15330348
F_K326_S3_Leaf_1