Assessing Fire Survival Potential via Soil Microbiome

To assess fire survival potential, RNA and DNA were extracted from soil collected at 24 h post-burn using the RNeasy PowerSoil Total RNA kits and RNeasy PowerSoil DNA Elution kits (QIAGEN) following manufacturer instructions. Residual DNA contamination was removed from the RNA extracts using DNase Max kits (QIAGEN) following manufacturer instructions. RNA reverse transcription was carried out using Invitrogen SuperScript IV VILO Master Mix (ThermoFisher) following manufacturer instructions. Total RNA and DNA were quantified using a Quant-iT RiboGreen RNA Assay kit (ThermoFisher) and Invitrogen Quant-iT PicoGreen dsDNA Assay kits (ThermoFisher), respectively. For the fast growth and post-fire affinity incubations, DNA was extracted from soil collected at the end of the incubations using the DNeasy PowerLyzer PowerSoil DNA Extraction kit following manufacturer instructions.
Copy DNA and DNA were amplified via triplicate PCR, targeting the 16S rRNA gene v4 region of bacteria and archaea with 515f and 806r primers^{74 (link)}, with barcodes and Illumina sequencing adapters added following ref. ^{75 (link)} (all primers are listed in Supplementary Tables 15 and 16). PCR amplicon triplicates were pooled, purified, normalized and sequenced using 2 × 250 paired-end Illumina MiSeq sequencing at the UW-Madison Biotechnology Center.