Flex library prep kit

RNA was extracted from liquid culture using the Zymo Fungal/Bacterial RNA MiniPrep Kit. Using the NEBNext Poly(A) mRNA Magnetic Isolation Module, polyA tailed mRNA was isolated from the total RNA. Two ONT direct RNA sequencing libraries were prepared and sequenced on separate MinION flowcells, each using ∼200 ng of polyA selected RNA and the SQK-RNA002 sequencing kit. With the NEBNext Ultra II RNA First-Strand Synthesis Module and the NEBNext Ultra II Non-Directional RNA Second Strand Synthesis Module, cDNA was prepared from the isolated mRNA. Two individual Illumina libraries were then prepared with the Nextera Flex Library Prep Kit, each using 400 ng of cDNA. Both library replicates were then sequenced on a single iSeq 100 run, generating 2 × 150 paired-end reads.

Genomic DNA was isolated from overnight LB cultures using the GeneJET genomic DNA purification kit (Thermo Fisher Scientific). A 150-bp paired-end library was prepared using the Flex library prep kit (Illumina) and the Nextera DNA CD index kit (Illumina). Whole-genome sequencing of mutants was performed on an Illumina MiniSeq sequencer. Mutation analysis was performed using CLC Genomics Workbench. The sequencing reads were trimmed and mapped to the reference genome (MG1655) and analyzed for single nucleotide polymorphisms (SNPs), indels, and structural variants. Detected mutations were subsequently confirmed by Sanger sequencing.

Genomic DNA was isolated from overnight LB cultures using the GeneJET genomic DNA purification kit (Thermo Fisher Scientific, Waltham, MA, USA), after which 150- bp paired-end libraries were prepared using the Flex library prep kit (Illumina, San Diego, CA, USA) and the Nextra DNA CD index kit (Illumina). Sequencing was performed with an Illumina MiniSeq sequencer and analyzed with CLC Genomics Workbench (Qiagen, Hilden, Germany). The sequencing reads were trimmed and mapped to the reference genome (MG1655) and analyzed for single nucleotide polymorphisms (SNPs), indels, and structural variants.