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.  Genomic DNA was extracted from mycelia using both the CTAB method and a protocol recommended by the manufacturer (User-Developed Protocol: Isolation of genomic DNA from plants and filamentous fungi using the QIAGEN® Genomic-tip, Qiagen Inc.). RNA was extracted from fungal isolates and from tobacco detached leaves infected with corresponding fungal isolates, using the Qiagen RNeasy Plant Mini Kit (Qiagen Inc. Germantown, MD, USA). Extracted genomic DNA and RNA was quantified with a Qubit Flex Fluorometer (Thermo Fisher Scientific, Waltham, MA, USA). AG and subgroup identity of the fungal isolates was verified by ITS-PCR, sequencing and homology analysis with nucleotide sequences available in the NCBI database. For making genomic libraries, an input of 500ng of DNA from each sample was sheared on Covaris (Covaries E series) and paired-end libraries were prepared for sequencing using Illumina's HiSeq 2000 platform. From end repair until adapter ligation and purification steps of the paired-end libraries were prepared using the protocol \"Illumina library prep\" on the IP-Star automated platform from Diagenonde (Diagenode IP Star) as per the manufacturer's protocol. Post ligation, manual protocols were used for gel size selection and PCR amplification using the standard Illumina PCR Cycle (Kapa high-fidelity master mix). The prepared libraries were analyzed on bioanalyzer and quantified using Qubit (Thermo Fisher). The normalized libraries were pooled for sequencing (insert size of 500bp) and submitted for HiSeq 2000 sequencing at Bioscience Core Laboratory of King Abdullah University of Science and Technology.