Soil fecal rna kit

For RNA isolation 2 mL of reactor liquid samples were used. The samples were centrifuged at 12,000 rpm for 10 min. 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™ DNA removal kit (K2981, Thermo Fisher Scientific, Waltham, MA, United States). Ribosomal RNA was depleted using the Ribo-Zero™ rRNA Removal Kit for Bacteria (Illumina, Madison, USA) according to the manufacturer’s instructions. The rRNA depleted samples were purified via the RNA Clean and Concentrator Columns from Zymo Research (Irvine, USA). During this step, an additional in-column DNase I treatment was included to ensure complete removal of DNA. Subsequently, synthesis of double-stranded cDNA was conducted using the Maxima H Minus Double-Stranded cDNA Synthesis Kit from ThermoScientific (Waltham, USA). In the first-strand cDNA synthesis reaction, 2 μL of random hexamer primer were used. Final purification of the blunt-end double-stranded cDNA was carried out using SureClean Plus solution from Bioline (Luckenwalde, Germany). The cDNA was sequenced in the same way as the total DNA. The quality of the RNA preparation was checked by agarose gel electrophoresis (data not shown).

The RNA extractions were carried out with the Zymo Research Soil/Fecal RNA kit (R2040, Zymo Research, Irvine, CA, USA). After lysis (bead beating), the Zymo Research kit protocol was followed. The DNA contamination was removed by Thermo Scientific Rapid Out™ DNA removal kit (K2981, Thermo Fisher Scientific, Waltham, MA, USA). Before transcriptome sequencing, rRNA was depleted from RNA by using the Gram + /Gram − depletion kit in a 60:40 ratio (RiboMinus A15020 Life Technologies, San Francisco, CA, USA). The mRNA library was prepared using the mRNA Sample Prep kit (Illumina, San Diego, CA, USA). Sequencing was performed using the Illumina V2 chemistry (2 × 250 bp) and applying the MiSeq paired-end mode. Raw sequences are available on the NCBI Sequence Read Archive (SRA) under the accession number: PRJNA922065.

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).