RNA-seq of Geobacillus stearothermophilus
Illumina sequencing libraries were generated using the NEBNext Small RNA Library Prep kit with modifications as follows. Total RNA was diluted in FastAP buffer (Thermo Fisher Scientific) supplemented with SUPERaseIn RNase Inhibitor (Thermo Fisher Scientific) and fragmented by incubating at 92C for 1.5 minutes. The fragmented RNA was then DNase treated and dephosphorylated with TURBO DNase (Thermo Fisher Scientific) and FastAP (Thermo Fisher Scientific). Next, 5 end phosphorylation and 3 end repair were carried out using T4 PNK (NEB) in 1X T4 DNA ligase buffer (NEB). The RNA was column-purified using RNA Clean & Concentrator-5 (Zymo) and the concentration was determined using the DeNovix RNA Assay. Illumina adapter ligation and cDNA synthesis were performed using the NEBNext Small RNA Library Prep kit, with substitution of a custom i7 adapter containing a 10-mer unique molecular identifier (UMI). Dual index barcodes were added by PCR amplification (10 cycles), and the cDNA libraries were purified using the Monarch PCR & DNA Cleanup Kit (NEB). High-throughput sequencing was performed on an Illumina MiniSeq in paired-end mode with 75 cycles per end with automated demultiplexing and adapter trimming.