Rumen Microbial Community DNA and RNA Isolation

For total community DNA isolation, 2 mL of rumen liquid samples were used. DNA extractions were carried out using a slightly modified version of the Zymo Research Fecal DNA kit (D6010, Zymo Research, Irvine, CA, United States). The lysis mixture contained 100 μL CTAB (cetyltrimethylammonium bromide) to improve the efficiency (Wirth et al., 2015a (link),b (link)). After lysis (bead beating), the Zymo Research kit protocol was followed.
For total RNA isolation, 1 mL of rumen liquid samples were taken. The RNA extractions were carried out with the Zymo Research Soil/Fecal RNA kit (R2040, Zymo Research, Irvine, CA, United States). After lysis (bead beating), the Zymo Research kit protocol was followed. The DNA contamination was removed by Thermo Scientific Rapidout^TM DNA removal kit (K2981, Thermo Fisher Scientific, Waltham, MA, United States).
The quantity of DNA and RNA was determined in a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE, United States) and a Qubit 2.0 Fluorometer (Life Technologies, Carlsbad, CA, United States). DNA purity was tested by agarose gel electrophoresis and on an Agilent 2200 TapeStation instrument (Agilent Technologies, Santa Clara, CA, United States). The quality of the RNA preparation was checked by agarose gel electrophoresis (data not shown).