GSM9437688: G217B DMSO RT day 2 C; Histoplasma ohiense; RNA-Seq
Total RNA was extracted from fungal cells as previously described [18-19] with Qiazol (Qiagen 79306). Frozen cell pellets were resuspended in equal volumes of Qiazol and subjugated to bead beating with 0.5 µM zirconia beads (Spectrum Chemical 140-19990-E1) and extraction using chloroform. The resulting aqueous phase was transferred to Epoch SpinTM RNA columns (Epoch Life Sciences 1940-250) to isolate RNA and subjected to sequential washes with 3 M NaOAc (pH = 5.5) and 10 mM TrisCl (pH = 7.5) in 80 % ethanol. The columns were then treated with Purelink DNAse (Invitrogen 12185010) and subjected to additional washes with 3 M NaOAc (pH = 5.5) and 10 mM TrisCl (pH = 7.5) in 80 % ethanol before being eluted with RNAse-free water. The organic phases of each sample were reserved at -20 °C for total protein extraction.  Equal amounts of total RNA (between 5-10 ng) across all samples were used for mRNA isolation. These samples were subjected to a second round of DNAse treatment (Turbo DNAse, Fisher Scientific AM1907) and their quality was assessed with an RNA 6000 Nano Bioanalyzer Kit (Agilent Technologies 5067-1511). Three rounds of poly-A selection using oligo-dT Dynabeads® (Invitrogen 61005) was used to purify mRNA, and mRNA enrichment was verified with an RNA 6000 Nano Bioanalyzer chip. Poly-A-selected RNA was used to generate libraries with the NEB Next Ultra II Directional RNA Library Prep Kit for Illumina (NEB E7760S). Each sample was dual indexed with unique primers (UCSF Center For Advanced Technology (CAT)) compatible for multiplexed sequencing. Library quality and size was assessed with the Agilent High Sensitivity DNA Bioanalyzer Kit (Agilent Technologies 5067-4626) to ensure proper fragment size (~300-400 bp) and the absence of contaminating adapters. Final library concentrations were measured using a Qubit HS dsDNA assay kit (Life Technologies Q33230) and equal amounts were pooled into a final library for sequencing. Pooled libraries were used for paired-end sequencing using one NextSeq 2000 lane at the Chan-Zuckerberg Biohub.