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/µL.This protocol is a compiled by using the Qiagen RNeasy® PowerSoil® Total RNA Kit with additional steps according the DNeasy Max®Kit (Qiagen), NEBNext® Ultra II Directional RNA Library Prep Kit for Illumina® (New England Biolabs, Inc), KAPA Library Quantification Kit for Illumina® Platforms (Kapa Biosystems), Qubit 2.0 (Thermo Fisher Scientific, Hvidovre, Denmark), Bioanalyzer® (Agilent Technologies, Inc.) sample Purification Beads (New England Biolabs) and Liquid G2 DNA/RNA Enhancer (Ampliqon) manufacturer protocols.