Expression profiling by high throughput sequencing
Summary
We tested a number of rRNA removal methods (Illumina RiboZero Plus, NEBNext, NEB Core Depletion Set with custom probes, siTools Panarchaea, siTools RiboPool) on 4 model halophile species: Halobacterium salinarum, Haloferax volcanii, Haloferax meditteranei, Haloarcula hispanica). It was found that methods using custom probes (NEB Core Depletion set with HVO probes, siTools RiboPool with HVO probes) efficiently remove rRNA in species they are targeted to, and that Panarchaea efficiently removes rRNA in all 4 tested species.
Overall design
Cells were grown to mid-exponential phase and cell pellets were extracted and immediately frozen in liquid N2. After 1-7 days at -80C, total RNA was extracted from frozen cell pellets using the Absolutely RNA Miniprep kit made by Agilent, with additional DNase treatment as necessary. 1-10ng of total RNA was used as input for rRNA removal methods, for which we followed manufacturer's protocols (with the exception of longer RNase digestion of 120 minutes instead of 30 minutes, for some samples of HBT treated with NEB Core Depletion set, labeled NEB_HVO_120). dsDNA libraries were created using NEBMultiplex Oligos (part of the NEBNext protocol for the relevant samples). They were quantified and quality-checked using DNA High Sensitivity Chip for the Agilent Bioanalyzer and were sent for sequencing to the Duke Center for Genomic and Computational Biology, where they were sequenced on Illumina NovaSeq 6000.
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