GSM6733077: SG1 dbcAMP A-2 (SG1_0mM); Histoplasma ohiense (nom. inval.); RNA-Seq
Total RNA was extracted from fungal cells using a Qiazol-based RNA extraction protocol. Frozen, resuspended pellets of cells were incubated at RT for 5 minutes to thaw. The lysate was subjected to bead beating (Mini-Beadbeater, Biospec Products, Bartlesville, OK) followed by a chloroform extraction. The aqueous phase was then transferred to an Epoch RNA column where the filter was washed with 3 M NaOAc (pH=5.5) and then with 10 mM TrisCl (pH=7.5) in 80% EtOH. DNase (Purelink, Invitrogen, Carlsbad, CA) treatment was used to remove any residual DNA, and the filters were washed again with NaOAc and TrisCl before eluting the RNA in nuclease-free water.  For each experiment, mRNA was extracted from an equal amount of total RNA (up to 20 μg) of each sample. Total RNA samples and were treated with rDNase I (Ambion). RNA quality was determined with a RNA 6000 Nano Bioanalyzer chip (Agilent Technologies, Santa Clara, CA). mRNA was purified using polyA selection with Oligo-dT Dynabeads (Thermo Fisher, Waltham, MA) as described in the manufacturer's protocol. Ribosomal RNA depletion was confirmed with an RNA 6000 Nano Bioanalyzer chip. Libraries for RNAseq were prepared using the NEB Next Ultra II Directional RNA Library Prep Kit (New England Biolabs, Ipswich, MA). Individual libraries were uniquely barcoded with NEBNext Multiplex Oligos for Illumina sequencing platform (New England Biolabs, Ipswich, MA). Average fragment size and presence of excess adapter was determined with High Sensitivity DNA Bioanalyzer chip from Agilent Technologies (Santa Clara, CA). Libraries had an average fragment length of 300 to 500 bp. The concentration of the individual libraries was quantified using the KAPA library quantification kit. A total of 5 ng of each library was pooled into each final libraries and run on a High Sensitivity DNA Bioanalyzer chip to determine the average fragment size of the final pooled samples. The final libraries were submitted to the UCSF Center for Advanced Technology for sequencing on an Illumina HiSeq 4000 sequencer.