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RNA-seq data for "Transcriptional control of hgcAB by an ArsR-like regulator in Pseudodesulfovibrio mercurii ND132"

Published on by Caitlin Gionfriddo

Transcriptomics Methods

RNA sequencing was performed on clean, DNAse-treated RNA extracted from the batch culture and washed-cell Hg-methylation assays. For the washed-cell assays, only RNA from the control, AsIII, and AsV treatments were sent for RNA-seq. All treatments from the culture Hg methylation assays were sent for sequencing. Three biological replicates for each treatment were sequenced. Library prep and sequencing were performed by Genewiz (Azenta Life Sciences, NJ). Ribosomal RNA (rRNA) depletion was performed during library preparation using three probes from QIAGEN FastSelect rRNA 5S/16S/23S Kit (Qiagen, Hilden, Germany), respectively. RNA sequencing libraries were prepared using NEBNext Ultra II RNA Library Preparation Kit for Illumina by following the manufacturer’s recommendations (NEB, Ipswich, MA, USA). Briefly, enriched RNAs are fragmented for 15 minutes at 94 °C, then first and second strand cDNA are synthesized. cDNA fragments are end repaired and adenylated at 3’ends, and universal adapters are ligated to cDNA fragments, followed by index addition and library enrichment with limited cycle PCR. Sequencing libraries were validated using the Agilent Tapestation 4200 (Agilent Technologies, Palo Alto, CA, USA), and quantified using Qubit 2.0 Fluorometer (ThermoFisher Scientific, Waltham, MA, USA) as well as by quantitative PCR (KAPA Biosystems, Wilmington, MA, USA). Multiplexed and clustered sequencing libraries were sequenced using Illumina Hiseq 2x150bp paired-end configuration. Raw sequencing files were uploaded to KBase.Initial read quality was assessed with FastQC, followed by adapter and quality-based trimming with Trimmomatic, alignment to Pseudodesulfovibrio mercurii ND132 reference genome (https://www.ncbi.nlm.nih.gov/assembly/GCF_000189295.2) with HISAT2, assembly of transcripts from alignment using StringTie. Differential expression analysis was performed with DeSeq2 and the results were uploaded to Galaxy web platform, and we used the public server at usegalaxy.org to produce figures using the Galaxy R scripts.

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