Acute effects on the human peripheral blood transcriptome of decompression sickness secondary to scuba diving: Supplementary datasets

In this study, we aim to explore the evolution of leukocyte gene expression in human subjects with DCS compared to closely matched divers after uneventful diving using a hypothesis-free RNA sequencing approach .Recruitment of DCS cases (n = 7) and controls (n = 6) was carried out using specific criteria. For both DCS cases and controls, whole blood was sampled at two time points, T1 - within 8 hours of surfacing from diving and T2 - at 40-44 hours after surfacing. Prior to sampling at T2, subjects were fasted for 10 hours. Specific exclusion criteria included a) age < 18 years b) self-reported consumption of alcohol and/or strenuous physical activity before T2 c) symptoms suggestive of delayed DCS presentation in controls at T2 d) acute life-threatening clinical complications or death within 72 hours of surfacing. All cases received emergency HBO as per United States Navy Treatment Table Six between T1 and T2.For RNA isolation, 2.5mL of whole blood was collected in a PAXgene® Blood RNA Tube (PreAnalytiX, Qiagen/BD) from DCS cases and controls at both T1 and T2. The quality of RNA was evaluated by RNA Integrity Number (RIN) determination using the RNA6000 Nano protocol on an Agilent 2100 Bioanalyzer system (Agilent, USA). The RIN values for samples undergoing transcriptome analysis ranged from 7.8 to 9.3. Depletion of alpha and beta globin mRNA was carried out using the GLOBINclear™ kit (ThermoFisher Scientific). To minimize batch effects, all samples were processed simultaneously by the same investigator. RNA samples were submitted for library generation and sequencing by the Beijing Genomics Institute (BGI-Shenzhen). Briefly, poly(A) mRNA was enriched using poly(T) oligo-attached magnetic beads, followed by fragmentation. First strand cDNA synthesis was carried out using random hexamer N6 primers and reverse transcriptase. Following adaptor ligation to cDNA fragments, PCR amplification and purification, single stranded DNA circles were generated in a final library. DNA nanoballs (DNBs) were subsequently generated by rolling circle replication, which underwent paired end sequencing (100bp) on the BGI DNBseq platform本研究旨在采用无预设假说的RNA测序策略，探究减压病（Decompression Sickness, DCS）患者与经过严格匹配的对照潜水员在无并发症潜水后外周血白细胞基因表达的动态变化。本研究通过特定标准招募了7例减压病患者与6例对照潜水员。所有研究对象均于两个时间点采集外周全血样本：T1为潜水出水后8小时内，T2为出水后40~44小时。T2采样前，所有受试者需禁食10小时。本研究的具体排除标准如下：a) 年龄小于18周岁；b) T2采样前自述饮酒或进行过剧烈体力活动；c) 对照潜水员在T2采样时出现疑似迟发性减压病的症状；d) 出水后72小时内出现急性致命性临床并发症或死亡。所有减压病患者均于T1与T2采样间期，按照美国海军治疗表6号接受急诊高压氧（Hyperbaric Oxygen, HBO）治疗。在T1与T2两个时间点，分别采集2.5mL外周全血至PAXgene®血液RNA采血管（PreAnalytiX，Qiagen/BD）中，用于RNA提取。采用美国安捷伦2100生物分析仪系统，通过RNA6000 Nano试剂盒方案测定RNA完整性数值（RNA Integrity Number, RIN），以此评估RNA质量；用于转录组分析的样本其RIN值介于7.8~9.3之间。采用GLOBINclear™试剂盒（赛默飞世尔科技，ThermoFisher Scientific）去除α、β珠蛋白mRNA。为最大限度降低批次效应，所有样本均由同一名实验人员同步完成处理。随后将RNA样本送至深圳华大基因（Beijing Genomics Institute, BGI-Shenzhen）进行文库构建与测序。简要而言，测序文库构建流程如下：首先利用结合poly(T)寡核苷酸的磁珠富集poly(A)信使RNA（mRNA），随后对其进行片段化；采用随机六聚体N6引物与反转录酶合成第一条cDNA链；将衔接子连接至cDNA片段后，经过PCR扩增与纯化，最终生成单链DNA环状文库；通过滚环复制制备DNA纳米球（DNA nanoballs, DNBs），随后在BGI DNBseq平台上完成100bp双端测序。