RNA-Seq of Cryptococcus gattii VGII R265
Cells were grown in YPDmedium overnight, and 10e6 cells were recovered by centrifugation, washed three timesin sterile water, and stored at 80C. Cell lysis was performed by adding 750 L of Trizoland zirconium beads to the frozen pellet. Samples were agitated using the FastPrep-24(MP Biomedical) at 4.0 ms for 20 seconds and 1 min on ice in nine cycles. The mixturewas then incubated at room temperature for 5 min, and 200 L of iced chloroformwas added to each tube, which was hand shaken for 15 seconds, followed by a 3-minincubation at room temperature and a 12,000  g centrifugation at 4C for 15 min.The aqueous phase was transferred to a new tube, and 500 L of 100% isopropanolwas added, followed by a 10-min incubation at room temperature and centrifugationat 12,000  g for 10 min at 4C. The RNA pellet was washed with 1 mL of 75% coldethanol, vortex mixed, and centrifuged at 7,500  g for 5 min at 4C. Samples werevacuum dried and resuspended in PCR water for Nanodrop quantification, and the RNAquality was measured on the Agilent 2100 Bioanalyzer (Agilent Technologies, USA). TheRNA extracted from WT and mutant cells was prepared for RNA-Seq analysis accordingto the Illumina Stranded mRNA Prep Ligation protocol (Illumina, USA), following itsinstructions (Illumina, Document #1000000124518 v 02). The RNA sample input wasadjusted to a final concentration of 130 ng in distilled water with a final volume of25 L per well. Messenger RNA (mRNA) was captured using oligo (dT) magnetic beadsin a magnetic separation rack (New England Biolabs, Inc., USA), eluted in the elutionbuffer, and cleaned up using fragmentation master mix, bead binding buffer, and beadwashing buffer before fragmentation and denaturation of the mRNA using mixes andthermal cycles indicated by the manufacturer. To the purified mRNA, the first-strandsynthesis mix and the reverse transcriptase were added to synthesize the first cDNAstrand using the thermal cycler, followed by the synthesis of the second cDNA strand with the appropriate mix. After cDNA generation, the strands were cleaned up usingAgencourt AMPure XP and washing beads with fresh 80% ethanol. The 3 ends of thecDNA strands were adenylated with the A-Tailing Mix to prevent self-ligation betweenthe fragments and assure adapter complementarity by the thymine nucleotide locatedat the 3 end of the adapters. After adenylation, the samples were ligated to the indexanchors and PCR amplified. The fragments were captured with Agencourt AMPure XP,washed with 80% ethanol, and resuspended in resuspension buffer. The library was thenselectively amplified in an enhanced PCR mix for the anchor-ligated DNA fragments, andindexes and primer sequences were added to form clusters in a dual-indexed library.The library was finally checked for concentration and quality and then sequenced on theIllumina NextSeq 500 (Illumina, USA).