title: Illumina HiSeq 2000 paired end sequencing; The pangenome analysis of the soil-borne fungal phytopathogen Rhizoctonia solani and development of a comprehensive web resource: RsolaniDB
design: The pangenome analysis of the soil-borne fungal phytopathogen Rhizoctonia solani and development of a comprehensive web resource: RsolaniDB
library_construction_protocol: Fungal cultures were purified by the hyphal tip excision method and maintained by sub-culturing on potato dextrose agar (PDA, Sigma Aldrich catalog # P2182, St. Louis, MO, USA). The PDA was amended with kanamycin (25 µg/ml) and streptomycin (50 µg/ml) to inhibit bacterial growth. Isolates were grown in Potato Dextrose Broth (PDB, Sigma Aldrich catalog # P6685) broth at 100 rpm and 25 C for 4 to 6 days, mycelia collected by filtration through 2 layers of sterile cheese cloth, washed 2 X with sterile distilled water, gently squeezed and placed on 4 layers of paper towel to remove surface water, and then snap-frozen in liquid nitrogen and stored at -80 C till use.  Nicotina tabacum seedlings were raised to four-leaf stage on potting mix (Pro-mix, Premier Horticulture, USA) in the greenhouse at ambient temperature (22º - 24º C) and with four hours supplemental light with a mercury lamp. Two leaves were excised from each seedling and placed on a tray on two piece of wet paper towels. For inoculation, seven to eight agar plugs from the margin of fresh R. solani growth on 1/4th concentration of PDA (potato dextrose agar) were placed on the adaxial surface of each leaf. For control, only seven to eight agar plugs from 1/4th PDA were placed. Each tray was closed with a lid and incubated on lab bench at ambient temperature and light. After 5 days, yellow to necrotic symptoms were noticeable on R. solani treated leaves but no symptoms appeared on control leaves surrounding the plugs. The control and infected patches were excised with a sterile scalpel, snap frozen in liquid nitrogen and processed for RNA extraction with RNeasy Plus Mini Kit in RLC buffer (Qiagen Sciences Inc., Germantown, MD, USA). The purified RNA was treated with DNase at 37º C for 30 min, extracted with phenol and Phenol: chloroform, precipitated with ethanol, and dissolved in RNase-free water.  Strand-specific mRNA sequencing was performed from total RNA using TruSeq Stranded mRNA Sample Prep Kit LT (Illumina) according to manufacturer's instructions. Briefly, polyA+ mRNA was purified from total RNA using oligo-dT dynabead selection. First strand cDNA was synthesised using randomly primed oligos followed by second strand synthesis where dUTPs were incorporated to achieve strand-specificity. The cDNA was adapter-ligated and the libraries amplified by PCR. Libraries were sequenced in Illumina Hiseq2000 with paired-end 100bp read chemistry.