title: transcriptome of Barbatia virescens : foot tissue
design: Total RNA concentration was calculated by Quant-IT RiboGreen (#R11490, Invitrogen, Lofer, Austria)). The TapeStation RNA screentape (#5067-5576, Agilent Technologies, Palo Alto, CA, USA) is used to assess RNA integrity. A RIN greater than 7.0 was used only for RNA library construction. A library was independently prepared with 0.5ug of total RNA for each sample by Illumina TruSeq Stranded Total RNA Library Prep Gold Kit (#20020599, Illumina, Inc., San Diego, CA, USA). As part of the workflow, rRNA in the total RNA is removed. After this step, divalent cations are used to fragment the remaining mRNA. The cleaved RNA fragments are copied into first strand cDNA using SuperScript II reverse transcriptase (#18064014, Invitrogen, Lofer, Austria) and random primers. This is followed by second-strand cDNA synthesis using DNA Polymerase I, RNase H and dUTP. These cDNA fragments then go through an end repair process, the addition of a single A base, and then ligation of the adapters. Finally, PCR is used to enrich the cDNA library with purified products. The libraries were quantified using KAPA Library Quantification kits for Illumina Sequencing platforms according to the qPCR Quantification Protocol Guide (#KK4854, KAPA BIOSYSTEMS, Woburn, MA, USA) and qualified using the TapeStation D1000 ScreenTape (# 5067-5582, Agilent Technologies, Palo Alto, CA, USA). Indexed libraries were then submitted to an Illumina NovaSeq (Illumina, Inc., San Diego, CA, USA), and the paired-end (2100 bp) sequencing was performed by the Macrogen Incorporated. After preprocessing the raw reads from the sequencer, we assembled the processed reads using Trinity (trinityrnaseq_r20140717) with strand specific option: --SS_lib_type RF . The Trinity program was utilized for de novo transcriptome assembly, combining read sequences of a certain length of overlap to form longer fragments without N gaps, called unigenes. These unigenes were further processed for read alignment and abundance estimation with Bowtie v1.1.2 and RSEM v1.3.1.