GSM7743673: Amb 24h 3; Cryptococcus neoformans; RNA-Seq
Cells were collected via centrifugation from growth medium, lyophilized overnight and RNA harvested according to manufacturer protocol for Invitrogen PureLink RNA Mini Kit. Total RNA (>2 ug per sample) was submitted to Azenta Life Sciences for standard RNA-Seq next-generation sequencing. The RNA samples were quantified using Qubit 2.0 Fluorometer (ThermoFisher) and RNA integrity was checked using TapeStation (Agilent Technologies). The RNA sequencing libraries were prepared using the NEB Next Ultra II RNA Library Prep Kit for Illumina using manufacturer's instructions (New England Biolabs). Briefly, mRNAs were initially enriched with Oligod(T) beads. Enriched mRNAs were fragmented for 15 min at 94°C. First strand and second strand cDNA were subsequently synthesized. cDNA fragments were end repaired and adenylated at 3' ends, and universal adapters were ligated to cDNA fragments, followed by index addition and library enrichment by PCR with limited cycles. The sequencing libraries were validated on the Agilent TapeStation (Agilent Technologies) and quantified by using Qubit 2.0 Fluorometer (ThermoFisher) as well as by quantitative PCR (KAPA Biosystems). The sequencing libraries were multiplexed and clustered onto a flowcell. After clustering, the flowcell was loaded onto the Illumina HiSeq instrument according to manufacturer's instructions. The samples were sequenced using a 2x150 bp Paired End (PE) configuration. Image analysis and base calling were conducted by the HiSeq Control Software (HCS). Raw sequence data (.bcl files) generated from Illumina HiSeq was converted into fastq files and de-multiplexed using Illumina bcl2fastq 2.20 software. One mismatch was allowed for index sequence identification.