title: "Miscanthus lutarioriparius transcriptome project,the mature leaf sample of LZ6"
design: "From the same 14 populations of M. lutarioriparius, 40 individuals were sampled from each field site in this study.  Six individuals from four populations of M. sacchariflorus and four individuals representing four populations of M. sinensis were also sampled.  The fourth leaf from the top of each individual was cut and immediately placed in liquid nitrogen.  The samples were kept in liquid nitrogen and brought to the laboratory for RNA isolation.  Total RNA was isolated using Trizol reagent (Invitrogen) and puri?fied using the RNAeasy Mini RNA Kit (Qiagen).  The completeness of RNA was assessed by RIN (RNA Integrity Number) value (9.0) with an Agilent 2100 Bioanalyzer (Santa Clara, CA, USA).  The mRNA was isolated from purified total RNA using one round of purification with oligo d(T) beads (Dynabeads? mRNA Purification Kit (Invitrogen)).  The cDNA libraries were prepared with the NEBNext mRNA Library Prep Reagent Set for Illumina (New England BioLabs, Beverly, MA, USA).  First-strand cDNA was synthesized using random hexamer-primed reverse transcription.  Following second strand cDNA synthesis and adaptor ligation, cDNA fragments of approximately 250 bp were iso?lated by gel electrophoresis.  cDNA fragments were amplified by 12 cycles of PCR and 100-bp paired-end sequencing was performed on an Illumina HiSeq 2000.   The quality control of raw reads was performed with FASTQC and FASTX that identified and trimmed the first 9 bases and the last 11 bases with unstable sequence content or Phred quality scores less than 20 (Q20).  The remaining 80-bp paired-end reads were then input into FASTX that further filtered out reads showing more than 3% of bases with quality less than Q20."