454 sequencing of Datisca glomerata root transcript fragment library
The protocol used was modified after Chomczynski P (1993) A reagent for the single-step simultaneous isolation of RNA, DNA and proteins from cell and tissue samples. Biotechniques 15:532-534, 536-537. Plant tissue ground in liquid N2 and then transferred to a 2 ml microcentrifuge tube containing 1 ml of pre-warmed extraction buffer (65 degrees C, 2 % CTAB, 2 % PVP (K30), 100 mM Tris-HCl (pH 8.0), 25 mM EDTA, 2.0 M NaCl, 0.5 g/l spermidine, 2 % beta-mercaptoethanol) per 200 mg of plant material, and mixed by inverting and vortexing. The mixture was extracted twice with an equal volume of (25:24:1) phenol:chloroform:isoamyl alcohol and once with (24:1) chloroform:isoamyl alcohol. Phase separation of phases was achieved by centrifugation at 10 krpm at room temperature for 10 min. The resulting RNA was precipitated using 1/10 volume of 3 M sodium acetate pH 5.2 and 2.5 volumes of absolute ethanol at -20 degrees C over night. Libraries were prepared according to Texeira Torres et al. (2007) Gene expression profiling by massively parallel sequencing. Genome Res. 2008 18: 172-177:  Methods for the library preparation were based on previously described SAGE (Velculescu et al. 1995) and GLGI (Generation of Longer cDNA fragments for Gene Identification; Chen et al. 2002) protocols. Double-stranded cDNA was digested separately with the following restriction endonucleases: MboI (Sau3AI, DpnII), NlaIII, and TaiI. 3' Fragments were recovered using M-270 Streptavidin beads (Dynal). After selection, specific linkers for each enzyme were ligated to the 3' fragments. The linkers consisted of the double-stranded 454 sequencing primer A (5'-GCCTCCCTCGCGCCATCAG-3') and a four-base overhang complementary to the enzyme restriction site. The three enzyme libraries were pooled before sequencing.