GSM7060407: Pistia Leaf 3; Pistia stratiotes; RNA-Seq
TRIZOL/Qiagen RNEasy RNA integrity was assessed using the RNA Nano 6000 Assay Kit on a Bioanalyzer 2100 (Agilent Technologies, CA, USA). Total RNA was used as input. mRNA was purified from total RNA using poly-T oligo-attached magnetic beads. Fragmentation was carried out using divalent cations under elevated temperature in First Strand Synthesis Reaction Buffer(5X). First strand cDNA was synthesized using random hexamer primer and M-MuLV Reverse Transcriptase (RNase H-). Second strand cDNA synthesis was subsequently performed using DNA Polymerase I and RNase H. Remaining overhangs were converted into blunt ends via exonuclease/polymerase activities. After adenylation of 3' ends of DNA fragments, adaptors with hairpin loop structure were ligated to prepare for hybridization. In order to preferentially select cDNA fragments of 370~420 bp in length, library fragments were purified with AMPure XP system (Beckman Coulter, Beverly, USA). Then PCR was performed with Phusion High-Fidelity DNA polymerase, Universal PCR primers and Index (X) Primer. PCR products were purified (AMPure XP system) and library quality was assessed on the Agilent Bioanalyzer 2100 system. Clustering of the index-coded samples was performed on a cBot Cluster Generation System using TruSeq PE Cluster Kit v3-cBot-HS (Illumia) according to manufacturer's instructions. After cluster generation, library preparations were sequenced on an Illumina Novaseq platform generating 150 bp paired-end reads