GSM5493410: Floral buds diameter less than 1.3cm of male flower 1; Handeliodendron bodinieri; RNA-Seq
Total RNA was isolated from samples using TRIzol reagent according to the manufacturer's protocol (Invitrogen). The first step in the workflow involves purifying the poly-A containing mRNA molecules using poly-T oligo-attached magnetic beads. Following purification, the mRNA is fragmented into small pieces using divalent cations under elevated temperature. The cleaved RNA fragments are copied into first strand cDNA using reverse transcriptase and random primers. This is followed by second strand cDNA synthesis using DNA Polymerase I and RNase H. These cDNA fragments then have the addition of a single 'A' base and subsequent ligationoftheadapter.Theproducts arethenpurifiedandenrichedwithPCR amplification. We then quantified the PCR yield by Qubit and pooled samples together to make a single strand DNA circle (ssDNA circle), which gave the final library. DNA nanoballs (DNBs) were generated with the ssDNA circle by rolling circle replication (RCR) to enlarge the fluorescent signals at the sequencing process. The DNBs were loaded into the patterned nanoarrays and pair-end reads of 100 bp were read through on the BGISEQ-500 platform for the following data analysis study. For this step, the BGISEQ-500 platform combines the DNA nanoball-based nanoarrays andstepwisesequencingusingCombinationalProbe-AnchorSynthesis SequencingMethod.