hPBDM-8-0hpi
RNA samples extracted from hPBDMs infected with T. marneffei PM1 were also subjected to RNASeq. Purities of the samples were measured using NanoDrop 1000 spectrometer (Thermo Fisher Scientific, Waltham, MA, USA). Purified RNA samples with 260 nm/280 nm ratio over 2.1 were then quantified by the fluorometric method using Qubit (Invitrogen). High-throughput Illumina sequencing was performed by the Centre for Genomic Sciences, Li Ka Shing Faculty of Medicine, The University of Hong Kong. Briefly, for each sample, a total of 700 ng of purified RNA were subjected to ribosomal RNA (rRNA) depletion using the Ribo-Zero Gold rRNA Removal Kit (Human/Mouse/Rat) (Illumina, San Diego, CA, USA). The cytoplasmic rRNA and mitochondrial rRNA were hybridised to biotinylated capture probes and then removed by washing. Subsequently, cDNA libraries were prepared using KAPA Stranded RNASeq Kit (KAPA Biosystems, Woburn, MA, USA) according to the manufacturers instructions. Theoretically, the rRNA-depleted RNAs were broken down into short fragments and used as templates for first-strand cDNA synthesis using random hexamer primer and reverse transcriptase. While during second-strand cDNA synthesis, the mRNA templates were removed, and replacement strands were generated to form blunt-end double-stranded (ds) cDNAs. These ds cDNAs were then subjected to 3 adenylation and indexed adaptor ligation. The adaptor-ligated libraries were enriched by 14 cycles of PCR. The enriched libraries were then denatured and diluted to optimal concentrations and used in the cluster generation steps. Finally, HiSeq PE Cluster Kit v4 with cbot (Illumina) was used for cluster generation on the flow cell, whereas HiSeq SBS Kit v4 (Illumina) was used for pair-end 101 bp sequencing.
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