GSM2061103: D-37C.CO2.90m-1-11.06.15-11.16.15-619; Cryptococcus neoformans; RNA-Seq
For RNA isolation, the culture was treated with ice-cold stop solution (5% Tris-saturated phenol in ethanol) to prevent RNA degradation and then collected by centrifugation. The cells were suspended in TRIzol reagent (Life Technologies) and lysed by mechanical bead-beating at 4°C with 0.5-mm silica-zirconia beads (4 cycles of 3 min of beating followed by 2 min on ice). Following lysis, total RNA was extracted according to the manufacturer’s instructions. Residual DNA was removed from the RNA preparation with the TURBO DNA-free kit (Life Technologies). Poly(A) RNA was selected from the total using the mRNA Catcher Plus Kit (Life Technologies) with an epMotion 5075 automated pipettor (Eppendorf). The poly(A) RNA was subsequently sheared by incubating in TURBO DNA-free buffer at 75°C for 10 minutes and purified with the QIAquick PCR Purification Kit (Qiagen). First strand cDNA synthesis was performed using random hexameric primers and SuperScript III Reverse Transcriptase, followed by treatment with E. coli DNA ligase, DNA polymerase I, and RNase H for second-strand synthesis, all using standard methods. The cDNA libraries were end-repaired with a Quick Blunting kit and A-tailed using Klenow exo- with dATP (New England Biolabs). Illumina adapters were ligated to the cDNA and fragments ranging from 150-250 bp in size were selected using gel electrophoresis. The libraries were enriched and indexed in a 10-cycle PCR using Phusion Hot Start II High-Fidelity DNA (Fermentas), purified, and pooled in equimolar ratios for multiplex sequencing on an Illumina HiSeq 2500 at the Genome Technology Access Center.
GEO
GSM2061103
GEO Accession
GSM2061103