title: RNA-seq of Medicago truncatula shoot (non-stressed)
design: Total RNA was isolated with the Spectrum Plant Total RNA Kit (Sigma) and then treated with TURBO DNA-free Kit (Invitrogen) to remove DNA molecules. RNeasy MinElute Cleanup Kit (Qiagen) was used to further clean the DNase-treated RNA samples. The quality of RNA samples was monitored by bio-analyzer analysis using Agilent RNA 6000 Nano Kit (Agilent). RNA samples were quantified using Qubit RNA BR (Broad-Range) Assay Kit (Life Technologies). RNA-seq libraries were prepared using TruSeq Stranded mRNA Sample Prep kits (Illumina). Individual libraries were uniquely indexed using TruSeq RNA Single Indexes (Illumina), and pooled in equimolar ratio.  The pooled libraries were sequenced on a Hiseq4000 system (Illumina). All sequences were first quality trimmed using a custom Perl script which removed low quality bases (quality score < 30).  Each sample was then de novo assembled with Trinity version 2.2.0.  These independent assemblies were then merged and aligned with HISAT2 version 2.0.5.  The aligned reads were assembled and quantified using Stringtie version 1.2.4.