RNA-seq of Tomato Leaves
1. Total RNA Extraction: Extract total RNA from tomato fruit samples using a commercial RNA extraction kit, and remove genomic DNA contamination with DNase I. 2. mRNA Purification: Enrich poly(A)-tailed mRNA from total RNA via magnetic beads conjugated with oligo(dT). 3. RNA Fragmentation: Break the purified mRNA into short fragments (200300 nt) with fragmentation buffer under high temperature. 4. cDNA Synthesis: Synthesize firststrand cDNA using random hexamer primers and reverse transcriptase; then generate doublestranded cDNA with DNA polymerase. 5. End Repair & Adapter Ligation: Repair the ends of doublestranded cDNA to blunt ends, add Atails to the 3 ends, and ligate with sequencing adapters. 6. Fragment Selection & PCR Amplification: Select targetsize cDNA fragments by agarose gel electrophoresis, and amplify the ligated products via PCR to construct the final transcriptome library. 7. Library Quality Control: Check library concentration, fragment size distribution, and purity using a Qubit fluorometer and Agilent 2100 Bioanalyzer.