Small RNA-seq and RNA-seq data

Small RNA-seqLibraries of small RNAs were constructed using TruSeq Small RNA Library Preparation Kits (Illumina) according to manufacturer’s protocols, and then sequenced by Illumina HiSeq 2000 at Bioacme (Wuhan, China). Quality control and adapter trimming process were performed using Trim Galore (v0.6.4) with default parameters, and reads length between 17 and 70 were used for subsequent analyses. The sequenced reads were aligned to the human genome (GRCh38) by using Bowtie2 (v2.3.5.1). Reads that cannot be aligned to the human genome (GRCh38) were then aligned to agshRNAs. The sequences and lengths of reads aligned to agshRNA were then determined using an in-house script. Reads mapped to genome were further categorized as miRNA, tRNA, snoRNA etc. by using htseq-count (v0.11.2). Annotation file was downloaded from DASHR 2.0 (https://dashr2.lisanwanglab.org/). RNA-seqLibraries of total RNAs were constructed using MGIEasy RNA Library Preparation Kits (BGI) according to manufacturer’s protocols, and then sequenced by MGISEQ2000 (BGI) at Wuhan Institute of Virology, CAS. Quality control and adapter trimming were performed using Trim Galore (v0.6.4) with default parameter. All reads were mapped to the human genome (GRCh38) using Hisat2 (v2.1.0) and then annotated using htseq-count (v0.11.2). Annotation file was downloaded from NCBI. Annotated reads number were transformed into count per million reads (CPM). Statistical significance was evaluated via using unpaired t test and adjusted using Bonferroni correction. Different expression genes (DEGs) were defined by |log2FC| > 1 and P.adj < 0.05.

小RNA测序（Small RNA-seq）文库构建：采用Illumina公司的TruSeq小RNA文库制备试剂盒，严格遵循制造商提供的官方实验方案构建小RNA文库，随后交由中国武汉Bioacme公司使用Illumina HiSeq 2000测序平台完成高通量测序。质控及接头修剪流程采用Trim Galore（v0.6.4）默认参数运行，仅保留长度介于17~70碱基的读段用于后续生物信息学分析。将测序读段比对至人类参考基因组GRCh38，比对工具为Bowtie2（v2.3.5.1）。无法比对至人类参考基因组GRCh38的读段，进一步比对至agshRNAs序列。利用实验室自研脚本确定比对至agshRNA的读段的序列与长度。比对至基因组的读段通过htseq-count（v0.11.2）进一步分类为微小RNA（miRNA）、转运RNA（tRNA）、核仁小RNA（snoRNA）等类别。注释文件从DASHR 2.0数据库（https://dashr2.lisanwanglab.org/）下载获取。
总RNA测序（RNA-seq）文库构建：采用BGI公司的MGIEasy RNA文库制备试剂盒，严格遵循制造商提供的官方实验方案构建总RNA文库，随后交由中国科学院武汉病毒研究所使用MGISEQ2000（BGI）测序平台完成高通量测序。质控及接头修剪流程采用Trim Galore（v0.6.4）默认参数运行。所有测序读段通过Hisat2（v2.1.0）比对至人类参考基因组GRCh38，随后通过htseq-count（v0.11.2）完成基因注释。注释文件从NCBI数据库下载。将注释后的读段计数转换为每百万读段计数（CPM，count per million reads）。统计学显著性采用非配对t检验进行评估，并通过Bonferroni校正进行多重检验校正。差异表达基因（DEGs）的筛选标准为|log₂FC| > 1且校正后P值（P.adj）< 0.05。