Abundant and diverse non-coding small RNAs identified in an extremophilic microbial community using metatranscriptomics

Regulatory small RNAs (sRNAs) represent a major class of regulatory molecules that play large-scale and essential roles in many cellular processes across all domains of life. Microbial sRNAs have been primarily investigated in a few model organisms and little is known about the dynamics of sRNA synthesis in natural environments, and the roles of these short transcripts at the community level. Analyzing the metatranscriptome of a model extremophilic community inhabiting halite nodules (salt rocks) from the Atacama Desert, sampled over two years with different weather conditions, with SnapT – a new sRNA annotation pipeline – we discovered hundreds of intergenic (itsRNAs) and antisense (asRNAs) sRNAs expressed. We sequenced metatranscriptomic libraries of halite nodules from the Atacama Desert over two sampling timepoints (2016 & 2017) to investigate the transcriptional response and sRNAs in the microbial community. Halite nodules were harvested in Salar Grande, an ancient evaporated lake in the Northern part of the Atacama Desert {Robinson, 2015 #6954} in February 2016 and 2017, 3 and 15 months after a major rain event (Uritskiy et al, 2019). All nodules were harvested within a 50m2 area as previously described {Robinson, 2015 #6954}. The colonization zone of each nodule was grounded into a powder, pooling from 1-3 nodules until sufficient material was collected, and stored in the dark in dry conditions until DNA extraction in the lab. Samples used for RNA were stored in RNAlater at 4°C until RNA extraction in the lab. Total RNA was extracted from the fixed samples by first isolating the cells through gradual dissolving of the salt particles as previously described {Crits-Christoph, 2016 #8776; Robinson, 2015 #6954} and lysing them through mechanical bead beating with the RNAeasy PowerSoil RNA extraction kit (QIAGEN). Total RNA was then extracted from the lysate with a Quick-RNA miniprep kit (Zymo Research). Total RNAseq libraries were prepared with the SMARTer Stranded RNA-seq kit (TaKaRa), using 25ng of RNA input and 12 cycles for library amplification. We sequenced 22 libraries from replicate samples from 2016 and 24 libraries from replicate samples from 2017.