Transcriptome analysis of high humidity-regulated genes in Arabidopsis thaliana

Arabidopsis Col0 were grown in pots for 12 days before the transfer to a humidity-controlled chamber set at relative humidity 30% for additional 2 days. High humidity treatments were performed for 0, 10-min and 30 min in a chamber with high humidity (higher than 90%). Triplicate experiments were performed for each time point.Total RNA was isolated using the Qiagen RNeasy Plant Mini Kit. The RNA was then treated with TURBO DNase (Thermo Fisher Scientific). The quality of DNA-free RNA was assessed using Bioanalyzer 2100 (Agilent, Palo Alto, CA, USA). PolyA selection was conducted using Dynabeads mRNA purification Kit (Thermo Fisher Scientific, Waltham, MA, USA) to isolate mRNA, followed by shearing to approximately 300-base fragments in size using Covaris S2 Ultrasonicator (Covaris, Woburm, MA, USA). RNA-seq libraries were constructed using MGIEasy RNA directional library prep kit (Complete Genomics Inc, San Jose, CA, USA). For RNA-seq sequencing, samples were run multiplexed on the MGI DNBSEQ-G400RS FCL flowcell at 100-bp PE length.

将拟南芥Col0生态型（Arabidopsis Col0）种植于盆中12天，随后转移至相对湿度设置为30%的控湿培养箱中继续培养2天。将材料置于相对湿度高于90%的培养箱中开展高湿度处理，处理时长分别为0分钟、10分钟及30分钟。每个时间点均设置三次生物学重复。总RNA提取采用Qiagen RNeasy植物小型试剂盒（Qiagen RNeasy Plant Mini Kit），随后使用TURBO DNase（Thermo Fisher Scientific）处理RNA以去除基因组DNA。利用生物分析仪2100（Bioanalyzer 2100，Agilent，美国加利福尼亚州帕洛阿尔托）评估无DNA RNA的质量。采用Dynabeads mRNA纯化试剂盒（Dynabeads mRNA Purification Kit，Thermo Fisher Scientific，美国马萨诸塞州沃尔瑟姆）进行polyA富集以分离mRNA，随后使用Covaris S2超声波破碎仪（Covaris S2 Ultrasonicator，Covaris，美国马萨诸塞州沃本）将mRNA破碎为约300个碱基对的片段。RNA测序文库构建采用MGIEasy RNA定向文库制备试剂盒（MGIEasy RNA Directional Library Prep Kit，Complete Genomics Inc，美国加利福尼亚州圣何塞）。RNA测序阶段，样本采用MGI DNBSEQ-G400RS FCL流动槽进行100碱基对双端的多重测序。