RNA extraction & depletion v1

This protocol provides a detailed methodology for the extraction and purification of RNA from both enrichment cultures and sampels from anaerobic digesters. The targeted microbial communities include organisms relevant to the anaerobic digestion process. Protocol overview: 1. Sample preparation Protocol includes description for; a) Samples from enrichment/pure cultures. b) Samples from anaerobic digesters. 2. RNA extraction & purification RNA is extracted using TriZol/Chloroform extraction. Extraction is followed by purification using Quick-RNA™ Fecal/Soil Microbe Microprep kit, excluding the cell lysis and extraction step included in the kit. Kit protocol is followed from step 4. 3. Quality control Agilent 2100 BioAnalyzer used for quality control and concentration measurement. The assay "Prokaryote Total RNA Nano Series II" is used at this step. 4. rRNA depletion Ribosomal RNA is removed using the Pan-Prokaryote riboPOOL kit, followed by ethanol precipitation. 5. Concentration measurement Agilent 2100 BioAnalyzer used for quality control and concentration measurement. The assay "mRNA Nano Series II" is used at this step. It is recommended that Sample preparation, RNA extraction & purification, and Quality control is carried out without pause. After the quality control, samples can be stored at -80ºC until further processing.