RNA-Seq of Physalis philadelphica L:flower
Isolate and purify total RNA from samples using TRIzol (Thermo Fisher, 15596018) according to the manufacturer's protocol. Then, perform quality control of total RNA quantity and purity using NanoDrop ND-1000 (NanoDrop, Wilmington, DE, USA) and assess RNA integrity using Bioanalyzer 2100 (Agilent, CA, USA). Ensure concentration >50 ng/L, RIN value >7.0, and total RNA >1 g to meet downstream experimental requirements. Employ oligo(dT) magnetic beads (Dynabeads Oligo (dT), cat.25-61005, Thermo Fisher, USA) for specific capture of mRNA containing PolyA tails through two rounds of purification. Fragment the captured mRNA under high-temperature conditions using a magnesium ion disruption kit (NEBNextR Magnesium RNA Fragmentation Module, cat.E6150S, USA) at 94C for 5-7 minutes. Synthesize cDNA from fragmented RNA using Invitrogen SuperScriptTM II Reverse Transcriptase (cat.1896649, CA, USA). Conduct double-stranded synthesis with E. coli DNA polymerase I (NEB, cat.m0209, USA) and RNase H (NEB, cat.m0297, USA) to convert the DNA-RNA hybrid double strands into DNA double strands, while incorporating dUTP Solution (Thermo Fisher, cat.R0133, CA, USA) into the double strands to blunt the ends. Add an A base to each end of the strands to enable connection with adapter molecules containing T bases at the ends and use magnetic beads for size selection and purification. Digest the double strands with UDG enzyme (NEB, cat.m0280, MA, US), followed by PCR pre-denaturation at 95C for 3 minutes, denaturation at 98C for a total of 8 cycles with each cycle consisting of 15 seconds, annealing at 60C for 15 seconds, extension at 72C for 30 seconds, and a final extension at 72C for 5 minutes to create a library of fragments with a size of 300bp 50bp (strand-specific library). Finally, perform paired-end sequencing using the Illumina NovaseqTM 6000 (LC Bio Technology CO.,Ltd. Hangzhou, China) according to standard protocols, with a sequencing mode of PE150.