title: GSM1679375: H88 Yeast B; Histoplasma capsulatum; RNA-Seq
library_construction_protocol: Hyphal and yeast cells were collected by centrifugation or filtration and total RNA was isolated using a guanidine thiocyanate lysis protocol as previously described (Hwang et al, Molecular Biology of the Cell 14:2314). Paired-end RNA-seq libraries were made from biological duplicate cultures of G217B, G186AR, H88, and H143 for both yeast and hyphae. DNA was depleted from 10 – 20 μg of total RNA by 1 hour of DNaseI digestion at 37°C (Ambion by Life Technologies, Carlsbad, CA). Poly(A)+ RNA was selected from DNA-depleted RNA on oligo- dT25 DynaBeads (Invitrogen by Life Technologies) using 2 rounds of selection per the manufacturer’s instructions. Approximately 50 ng of poly(A)+ RNA was used as input for generating sequencing libraries with the ScriptSeq v2 RNA-Seq Library Preparation Kit (Epicentre, Madison, WI) per the manufacturer’s instructions with the following modifications.  After 11 cycles of the ScriptSeq v2 PCR cDNA amplification step, cDNA libraries were resolved on an 8% TBE gel (Invitrogen by Life Technologies) and a 350 – 500 bp range of cDNA library was excised, eluted, and precipitated from the gel. Seven additional PCR cycles were resumed following the ScriptSeq v2 Kit protocol. cDNA libraries were purified with AMPure XP beads (Beckman Coulter, Brea, CA) to deplete primers. Libraries were multiplexed and subjected to 100 bp paired-end sequencing using the Illumina HiSeq2000 sequencer (Illumina, San Diego, CA).
attributes.tag: GEO Accession
attributes.value: GSM1679375