Small RNA sequencing of Pongamia seeds
Total RNA was isolated from Pongamia seeds using a modified CTAB method. At each sampling time point, seeds from each of the three trees were separately subjected to RNA extraction. The resulting nine RNA samples were further purified with the RNeasy Plant Mini Kit. The RNA molecules with different sizes were separated using polyacrylamide gel electrophoresis. The 18-30 nt small RNAs were excised and then ligated to 5 and 3 adapters by T4 RNA ligase in two separate steps. The ligation products were reverse transcribed and then amplified by PCR. The 140-160 bp PCR products were enriched to generate nine cDNA libraries.