GSM3660308: QS-rde1-B; Cryptococcus neoformans var. grubii; RNA-Seq
genomic DNA: Cells were lyophilized then powdered by bead beating with 0.5 mm zirconia-silica beads. Genomic DNA was extracted as described in Chun and Madhani, Methods Enzym. 2010. PolyA mRNA, small RNA: Cells were lyophilized then powdered by bead beating with 0.5 mm zirconia-silica beads. RNA was then extracted using Trizol and chloroform. ATMT insertional sequencing: 20 μg of genomic DNA was sonicated for 10 min (30 sec on, 1 min rest) at 4°C. DNA was re-purified with phenol/chloroform/isoamyl alcohol (25:24:1, Sigma), washed with chloroform and precipitated with 3 volumes ethanol. Linear PCR originating in the T-DNA:NAT insertion was performed with Accuprime Taq High Fidelity polymerase (Thermo Fisher) using the JEBPN-Biotin2 primer. First strand DNA was then purified over M280 Dynabeads (Thermo Fisher) according to the manufacturer's instructions. Purified linearized DNA was ligated to the JEBPN-DNA linker using Circligase II (Epicentre) according to the manufacturer's instructions. Finally, DNA was amplified by nested PCR with JEBPN-SA-II and JEBPN_index-SA-I (Table S6) with various indexes using Accuprime Taq High Fidelity polymerase (Thermo Fisher). RNA-seq: NEBNext Ultra II Directional library prep kit for Illumina according to manufacturer's instructions. small RNA-seq: Lexogen Small RNA-seq library prep kit according to manufacturer's instructions. QuantSeq: Lexogen QuantSeq 3' mRNA library prep kit according to manufacturer's instructions.
GEO Accession
GSM3660308