RNA_Seq of Oxytenanthera abyssinica
Total RNA of each sample was extracted using TRIzol Reagent(Invitrogen)/RNeasy Mini Kit (Qiagen)/other kits. Total RNA of each sample wasquantified and qualified by Agilent 2100 Bioanalyzer (Agilent Technologies, PaloAlto, CA, USA), NanoDrop (Thermo Fisher Scientific Inc.) and 1% agrose gel. 1g total RNA with RIN value above 7 was used for following library preparation.Next generation sequencing library preparations were constructed according to themanufacturers protocol (NEBNext Ultra RNA Library Prep Kit forIllumina).The poly(A) mRNA isolation was performed using NEBNext Poly(A) mRNAMagnetic Isolation Module (NEB) or Ribo-Zero rRNA removal Kit (illumina).The mRNA fragmentation and priming was performed using NEBNext FirstStrand Synthesis Reaction Buffer and NEBNext Random Primers. First strandcDNA was synthesized using ProtoScript II Reverse Transcriptase and the secondstrand cDNA was synthesized using Second Strand Synthesis Enzyme Mix. Thepurified double-stranded cDNA by AxyPrep Mag PCR Clean-up (Axygen)wasthen treated with End Prep Enzyme Mix to repair both ends and add a dA-tailing inone reaction, followed by a T-A ligation to add adaptors to both ends. Sizeselection of Adaptor-ligated DNA was then performed using AxyPrep Mag PCRClean-up (Axygen), and fragments of ~360 bp (with the approximate insert size of300 bp) were recovered. Each sample was then amplified by PCR for 11 cyclesusing P5 and P7 primers, with both primers carrying sequences which can annealwith flow cell to perform bridge PCR and P7 primer carrying a six-base indexallowing for multiplexing. The PCR products were cleaned up using AxyPrep Mag PCR Clean-up (Axygen), validated using an Agilent 2100 Bioanalyzer (AgilentTechnologies, Palo Alto, CA, USA), and quantified by Qubit 2.0 Fluorometer(Invitrogen, Carlsbad, CA, USA).Then libraries with different indices were multiplexed and loaded on an IlluminaHiSeq instrument according to manufacturers instructions (Illumina, San Diego,CA, USA). Sequencing was carried out using a 2x150bp paired-end (PE)configuration; image analysis and base calling were conducted by the HiSeqControl Software (HCS) + OLB + GAPipeline-1.6 (Illumina) on the HiSeqinstrument.