alias: GSM3528228
title: GSM3528228: SG1 GlcNAc t=0d 3-A; Histoplasma capsulatum G217B; RNA-Seq
library_construction_protocol: Histoplasma capsulatum cultures were collected by filtration with a disposable filtration apparatus either in Biosafety Level 2 laboratory for growth at 37°C or in the Biosafety Level 3 laboratory for growth at room temperature. At each time point, 10mL of fungal culture was passed through a disposable filter apparatus (Thermo Fisher), the cells were scraped off with a cell scraper, placed into a conical with 1mL trizol reagent (Qiazol) and flash frozen in liquid nitrogen. Samples were stored at -80°C until all time points were collected.  Total RNA was extracted from fungal cells using a trizol-based RNA extraction protocol. Frozen, resuspended pellets of cells were incubated at room temperature for 5 minutes to thaw. The lysate was bead beat followed by a chloroform extraction. The aqueous phase was then transferred to an Epoch RNA column where the filter was washed with 3M NaOAc and then with 10mM TrisCl in 80% EtOH. DNase (Purelink) 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. RNA quality was determined with a High Sensitivity DNA bioanalyzer chip from Agilent.  20ug of total RNA was purified for mRNA through polyA selection with Oligo-dT Dynabeads(Thermo Fisher) as described in the Thermo Scientific protocol. Ribosomal RNA depletion was confirmed with an RNA 6000 nano bioanalyzer chip from Agilent Technologies. Samples were acceptable for library preparation at 0-4% rRNA. Libraries for RNAseq were prepared using the NEBNext Ultra Directional RNA Library Prep Kit (New England Biolabs). Individual libraries were uniquely barcoded with NEBNext Multiplex Oligos for Illumina sequencing platform (New England Biolabs). Average fragment size and presence of excess adapter was determined with High Sensitivity DNA bioanalyzer chip from Agilent Technologies. Libraries had an average fragment length of 300-500bp. The concentration of the individual libraries was quantified through Qubit dsDNA High Sensitivity Assay (Invitrogen). 5ng of each library was pooled into a final library and ran on a High Sensitivity DNA bioanalyzer chip to determine the average fragment size of the final pooled sample. Final library was submitted to the UCSF Center for Advanced Technology for sequencing on the Illumina HiSeq4000 sequencer.
identifiers.namespace: GEO
identifiers.id: GSM3528228