De novo transcriptome assembly of Calotropis procera leaves
Total RNA was isolated from young leaves of Calotropis procera using an RNeasy® Plant Mini Kit (QIAGEN, USA). The tissues were lysed in RLC buffer, and RNA was purified according to the manufacturer’s instructions. Isolated total RNA samples were treated with DNase to remove any contaminating genomic DNA. RNA concentrations were quantified using a NanoDrop spectrophotometer at a wavelength 260 nm. Integrity of the total RNA samples was evaluated using an Agilent 2100 Bioanalyzer (Agilent Technologies Inc., Santa Clara, CA), and samples with RNA integrity values above 8.3 were used in the experiments. Complementary DNA (cDNA) libraries were prepared from total RNA samples using an Illumina TruSeq RNA Sample Prep Kit (Illumina, San Diego, CA). One microgram of total RNA was used as the RNA input according to recommendations of the manufacturer’s protocol. Poly(A) mRNA was purified using oligo(dT)-conjugated magnetic beads and was eluted with Tris-HCl. Fragmentation medium was added to break the mRNA into short fragments. Taking these fragmented mRNAs as templates, random primers and SuperScript II reverse transcriptase (Invitrogen) were used to synthesize first-strand cDNA. This was followed by second-strand cDNA synthesis using DNA polymerase I and RNaseH. The ends of the resulting double-stranded cDNA molecules were repaired by adding a single ‘A’ base and then Illumina adapters were ligated to the repaired ends. cDNA fragments of 250-350 bp were purified from the gel and subjected to further template enrichment by PCR using two primers that anneal to the ends of the adapters to construct a fragmented cDNA library. The cDNA libraries were validated for RNA integrity and quantity using an Agilent 2100 Bioanalyzer (Agilent Technologies Inc.) before sequencing.