RNA-Seq of AIF 33
Soils were collected from field, passed through 4 mm sieve, amended with water (20% water content) and aircraft anti-icer fluid (3%, v/w) and incubated in dark for 15 d. Total RNA was immediately extracted from 1 g soil using a Qiagen RNeasy PowerSoil Total RNA Kit with additional treatment with Qiagen DNase Max to remove DNA contamination. Total RNA samples were immediately frozen, placed on dry ice, and sent FedEx priority overnight to UC Davis Genome Center for ribodepletion, library preparation, and sequencing. Strand-specific and dual-barcode indexed RNA-seq libraries were generated from 250 ng total RNA using the Kapa RNA-seq Hyper kit (Kapa Biosystems-Roche, Basel, Switzerland) and the QIAseq FastSelect5S/16S/23S ribodepletion reagent (Qiagen, Hilden, Germany) in combination following the manufacturers instructions. Fragment size distribution of the libraries was verified via micro-capillary gel electrophoresis on a LabChip GX system (PerkinElmer, Waltham, MA). Libraries were quantified by fluorometry on a Qubit instrument (Life Technologies, Carlsbad, CA) and pooled in equimolar ratios. The pool was quantified by qPCR with a Kapa Library Quant kit (Kapa Biosystems) and sequenced on an Illumina NovaSeq 6000 (Illumina, San Diego, CA) with paired-end 150 bp reads. The procedure generated 72+12 million raw reads per sample.