This experimental procedure was designed to isolate total the RNA aiming to obtain intact and high-quality integrity (RIN>7) for further next generation sequencing analysis. The protocol was developed to analyse the environmental microbial active community providing a clear overview of the microbial transcriptional activity in a specific time or after a different condition exposition sets, such as the investigation of thermal adaptation after heat-shock. The procedure is specifically designed to extract total-RNA from water samples although it can be used for various environmental samples as done in Schostag et al. and Bang-Andreasen et al. 2019 in soil with similar accuracy results. The total-RNA yield was >1000 ng\/mL with a RIN ranging from 7 to 8.5. The final Illumina library preparation yield ranged between 15 and 31 ng\/\u00b5L.This protocol is\u00a0a compiled by using the Qiagen RNeasy\u00ae PowerSoil\u00ae Total RNA Kit with additional steps according the DNeasy Max\u00aeKit (Qiagen), NEBNext\u00ae Ultra II Directional RNA Library Prep Kit for Illumina\u00ae (New England Biolabs, Inc), KAPA Library Quantification Kit for Illumina\u00ae Platforms (Kapa Biosystems), Qubit 2.0 (Thermo Fisher Scientific, Hvidovre, Denmark), Bioanalyzer\u00ae (Agilent Technologies, Inc.) sample Purification Beads (New England Biolabs) and Liquid G2 DNA\/RNA Enhancer (Ampliqon) manufacturer protocols.