small-RNA-Seq of P. brasiliensis (ATCC 32069) yeast phase supplemented with 10 M CuSO4 - rep2
P. brasiliensis (ATCC 32069 or Pb18) in the yeast phase was maintained on a solid Brain Heart Infusion (BHI) medium with 4% (w/v) glucose. After 3 days of growth on a solid medium, cells were cultured in liquid BHI medium under agitation at 150 rpm at 36C for 72 h. Then, 105 cells/mL were transferred to McVeigh/Morton minimal medium (MMcM) prepared without CuSO4 and supplemented with 50M of the copper chelator BCS (Sodium betocuproinedisulfonate - Sigma). For the control, the medium was supplemented with 10 M CuSO4. Treatment and control samples were prepared with three biological replicates. Cultures were maintained in agitation at 150 rpm for 24 h at 36C. Subsequently, cells were collected by centrifugation at 12,000 x g for 10 minutes at 4C. Then TRIzol (Life Technologies) was added for RNA extraction. RNA samples were obtained for each biological triplicates and stored at -80 C for sequencing.Library was prepared with NEBNext Small RNA RNA Library Prep Kit for Illumina (New England BioLabs Inc., Massachusetts, USA) following manufacturers instructions. Average final library size was 170 bp. Illumina 6-nt dual-indices were used for multiplexing. Samples were pooled and sequenced on Illumina NovaSeq S4 2x150 with a read length configuration of 150PE for 20M PE reads per sample (10M in each direction). Data was then trimmed to a read length of 1x50.