RNA-seq analysis dataset for Pseudomonas aeruginosa PAO1 37°C vs. 43°C

The data in this item includes raw RNA-sequencing data from post-43°C exposure and during the recovery period to assess transcript-level effect.Sample preparation: Overall, 18 samples included 3 replicates of Pseudomonas aeruginosa after exposure to 37 °C (control) or 43 °C (heat shock), at 3 time points (T=0hr, T=18hr, and T=54hr). Samples from the T=0 groups are simply labeled “37” or “43”. For RNA sequencing, 2 ug of total RNA was used for the RiboMinus™ Bacteria Transcriptome Isolation Kit (Invitrogen). The library was constructed with NEBNext® Ultra™ II Directional RNA Library Prep Kit for Illumina (NEB) according to the manufacturer's instructions using 30 ng of depleted RNA. The final quality was evaluated by TapeStation High Sensitivity D1000 Assay (Agilent Technologies, CA, USA). Sequencing was performed based on Qubit values and loaded onto an Illumina MiSeq using the MiSeq V2 (50- cycles) Kit (Illumina, CA, USA). Paired-end RNA-seq protocol was used, yielding about 3.4-6.5 million paired-end reads per sample. FastQC (v0.11.2) was used to assess the quality of raw reads.Analysis: Reads were aligned to Pseudomonas aeruginosa PAO1 strain (assembly GCF_000006765.1 ) using the bowtie2 aligner software (v2.3.2) with default parameters. GTF annotation file for the PAO1 strain was downloaded from NCBIPseudomonas Genome DB ( www.pseudomonas.com). Raw read counts for gene-level features were determined using HTSeq-count with the intersection-strict mode. Differentially expressed genes were determined with the R Bioconductor package DESeq2 (Release 3.14). The p-values were corrected with the Benjamini-Hochberg FDR procedure. Genes with adjusted p-values; 0.05.

本数据集包含43°C暴露后及其恢复期间原始RNA测序数据，旨在评估转录水平的影响。样本制备方面，总计18个样本，包括经37°C（对照组）或43°C（热休克）处理的铜绿假单胞菌的三份重复样本，分别在三个时间点（T=0小时、T=18小时和T=54小时）收集。T=0时间点的样本分别标记为“37”或“43”。RNA测序过程中，使用2ug总RNA与RiboMinus™细菌转录组分离试剂盒（Invitrogen）进行反应。根据制造商的说明，利用NEBNext® Ultra™ II定向RNA文库制备试剂盒（NEB）以及30ng去富集RNA构建文库。最终质量通过Agilent Technologies公司（CA，美国）的TapeStation高灵敏度D1000检测进行评估。测序基于Qubit值进行，并使用Illumina MiSeq测序仪以及MiSeq V2（50循环）试剂盒（Illumina，CA，美国）进行上样。采用双端RNA-seq协议，每个样本产生约3.4-6.5百万双端读取。使用FastQC（v0.11.2）软件评估原始读取质量。分析阶段，使用bowtie2软件（v2.3.2）以默认参数将读取与铜绿假单胞菌PAO1菌株（组装GCF_000006765.1）进行比对。从NCBIPseudomonas基因组数据库（www.pseudomonas.com）下载PAO1菌株的GTF注释文件。利用HTSeq-count软件在严格交集模式下确定基因水平特征的原读计数。差异表达基因通过R Bioconductor包DESeq2（Release 3.14）进行确定。p值通过Benjamini-Hochberg FDR方法进行校正。调整后的p值小于0.05的基因。