RNA-seq analysis dataset for <i>Pseudomonas aeruginosa </i>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.<b>Sample preparation: </b>Overall, 18 samples included 3 replicates of <i>Pseudomonas aeruginosa</i> after exposure to 37<i> </i>°C (control) or 43<i> </i>°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”.<b> </b>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.<b>Analysis</b>: Reads were aligned to <i>Pseudomonas aeruginosa </i>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.<br>

本数据集包含经43℃处理后及恢复期的原始RNA测序数据，用于评估转录本层面的效应。
样本制备：
总计18个样本，为铜绿假单胞菌（*Pseudomonas aeruginosa*）在37℃（对照组）或43℃（热激）条件下，于3个时间点（T=0hr、T=18hr、T=54hr）各设置3次生物学重复。T=0组的样本仅标记为"37"或"43"。
RNA测序环节中，取2μg总RNA使用RiboMinus™ 细菌转录组分离试剂盒（Invitrogen）进行核糖体RNA去除处理。随后以30ng经核糖体RNA去除后的RNA为起始材料，按照制造商说明书使用NEBNext® Ultra™ II 定向RNA文库制备试剂盒（NEB）构建适配Illumina平台的测序文库。最终文库质量通过TapeStation 高灵敏度D1000检测（安捷伦科技，美国加利福尼亚州）进行评估。测序前基于Qubit定量结果，使用MiSeq V2（50循环）试剂盒（Illumina，美国加利福尼亚州）将文库加载至Illumina MiSeq测序平台，采用双端RNA测序方案，每个样本可获得约340万至650万条双端reads。使用FastQC（v0.11.2）评估原始reads的质量。
数据分析：
使用bowtie2比对软件（v2.3.2，默认参数）将reads比对至铜绿假单胞菌PAO1菌株（组装编号GCF_000006765.1）。PAO1菌株的GTF注释文件从NCBI铜绿假单胞菌基因组数据库（www.pseudomonas.com）下载。使用HTSeq-count的交集严格模式计算基因层面的原始读长计数。使用R Bioconductor包DESeq2（版本3.14）鉴定差异表达基因，通过Benjamini-Hochberg FDR程序校正p值，筛选校正后p值<0.05的基因。

搜集汇总

背景与挑战

背景概述

该数据集包含铜绿假单胞菌PAO1在37°C（对照）和43°C（热休克）条件下的RNA-seq原始数据和分析结果，旨在评估热休克对转录水平的影响。实验设计包括18个样本，覆盖3个时间点（T=0hr, T=18hr, T=54hr），每个条件有3个生物重复，使用Illumina MiSeq平台进行配对末端测序。数据分析涉及基因比对、计数和差异表达分析，适用于研究细菌转录组响应热激的机制。