title: RNA-Seq  of algae: chlorella vulgaris
design: RNA quantification and qualificationRNA degradation and contamination was monitored on 1% agarose gels.RNA purity was checked using the NanoPhotometer spectrophotometer (IMPLEN, CA, USA).RNA concentration was measured using Qubit RNA Assay Kit in Qubit 2.0 Flurometer (LifeTechnologies, CA, USA).RNA integrity was assessed using the RNA Nano 6000 Assay Kit of the Agilent Bioanalyzer 2100system (Agilent Technologies, CA, USA). Library preparation for Transcriptome sequencingA total amount of 1.5 g RNA per sample was used as input material for the RNA samplepreparations. Sequencing libraries were generated using NEBNext Ultra RNA Library Prep Kitfor Illumina (NEB, USA) following manufacturers recommendations and index codes were addedto attribute sequences to each sample. Briefly, mRNA was purified from total RNA using poly-Toligo-attached magnetic beads. Fragmentation was carried out using divalent cations under elevatedtemperature in NEBNext First Strand Synthesis Reaction Buffer5X . First strand cDNA wassynthesized using random hexamer primer and M-MuLV Reverse TranscriptaseRNase H- . Secondstrand cDNA synthesis was subsequently performed using DNA Polymerase I and RNase H.Remaining overhangs were converted into blunt ends via exonuclease/polymerase activities. Afteradenylation of 3 ends of DNA fragments, NEBNext Adaptor with hairpin loop structure were ligatedto prepare for hybridization. In order to select cDNA fragments of preferentially 250~300 bp in length,the library fragments were purified with AMPure XP system (Beckman Coulter, Beverly, USA). Then3 l USER Enzyme (NEB, USA) was used with size-selected, adaptor-ligated cDNA at 37C for 15min followed by 5 min at 95 C before PCR. Then PCR was performed with Phusion High-FidelityDNA polymerase, Universal PCR primers and Index (X) Primer. At last, PCR products were purified(AMPure XP system) and library quality was assessed on the Agilent Bioanalyzer 2100 system. Clustering and sequencing (Novogene Experimental Department)The clustering of the index-coded samples was performed on a cBot Cluster Generation System usingTruSeq PE Cluster Kit v3-cBot-HS (Illumia) according to the manufacturers instructions. Aftercluster generation, the library preparations were sequenced on an Illumina Hiseq platform andpaired-end reads were generated.