title: Next-generation sequencing-based transcriptome profiling analysis of Pohlia nutans reveals insight into the stress-relevant genes in Antarctic moss
design: Frozen samples were ground in liquid nitrogen with mortar and pestle and then the ground powder was transferred into 1.5 ml eppendorf tubes. Total RNA was extracted from liquid nitrogen ground algae powder using TRIzol reagent (Invitrogen) following the standard protocol. The final total RNA was dissolved in 200 µL RNase-free water. The concentration of total RNA was determined by NanoDrop spectrophotometer (Thermo Scientific, USA), and the RNA integrity value (RIN) was checked using RNA 6000 Pico LabChip of Agilent 2100 Bioanalyzer (Agilent, USA).   Total RNA was incubated with 10 U DNase I (Ambion) at 37ºC for 1 hr, and then added Nuclease-free water to bring the sample volume to 250 µL. Messenger RNA was further purified with MicroPoly(A) PuristTM Kit (Ambion) following the manufacturer's manual. The mRNA was dissolve in 100 µL RNA Storage Solution and the final concentration was determined using NonoDrop spectrophotometer.  RNA was fragmented by 95ºC 5min and annealed with Biotinylated Random Primers which contained the Illumina adapter sequencing primer. Then the RNA fragments were captured by Streptavidin Magnetic Beads through interacting with Biotinylated Random Primers. Another Illumina adapter sequencing primer was added to 5' end of the cleaved mRNA fragments by T4 RNA ligase at 37ºC for 3hr. The cleaved RNA fragments were used for first strand cDNA synthesis using reverse transcriptase. The second strand cDNA were synthesized by using DNA polymerase I and RNaseH. Products were enriched and purified with PCR to create the final Illumina sequencing cDNA library.