Medicago sativa Gene Atlas 1.2
Total RNA was purified using RNeasy Plant Mini Kit (Qiagen, Valencia, CA, USA). Samples were sequenced by the University of Minnesota Genomics Center.  Poly-A RNA samples were isolated from total RNA using oligo-dT25 magnetic beads (Dynabeads; Invitrogen, Carlsbad, CA).  Poly-A RNA was denatured and used in a random-priming cDNA synthesis followed by end repair by T4 DNA polymerase, Klenow polymerase, and dNTPs.  End-repaired fragments are phosphorylated by T4 polynucleotide kinase followed by the addition of a single ‘A’ base to the 3’ end of the blunt end phosphorylated DNA fragments.  Illumina adapters were added to DNA fragments by ligation and size selected (500bp) by electrophoresis.  DNA libraries were amplified for 15 cycles.  Two picomoles of the cDNA library were loaded on an Illunina single-end flow cell using the Illunina Cluster Station (Illunina, Inc., San Diego, CA).  76bp reads were collected on an Illunina Genome Analyzer using sequencing by synthesis.  Sequenced reads were aligned to the MSGI 1.2 transcriptome using Bowtie2. Alignment files were sorted using Samtools.