Human benign adrenocortical tumors miRNome

Small RNAs (<100 bases in length) were purified from total RNA samples using the miRNeasy kit (Qiagen), and multiplexed miRNA libraries were prepared using a previously described protocol and sequenced on a HiSeq 2000 (Illumina). Image analysis, base calling, demultiplexing and conversion of BCL to FASTQ format were carried out using CASAVA 1.8.2 software (Illumina). Adaptor sequences were removed using mirExpress software. Fastq were 3p-adaptor trimmed. The sequences were then aligned on hg19 (GRCh37) human reference genome with STAR (v.2.5.2a), allowing no mismatch with reference. Reads were counted using HTSeq-count (HTSeq v 0.6.1p1 Python package) using “intersection-strict” mode, to count mature miRNA abundance according to miRbase v.20.

研究人员从总RNA样本中纯化得到长度小于100个碱基的小RNA（small RNAs），所用试剂盒为Qiagen公司的miRNeasy试剂盒（miRNeasy kit）；随后采用已报道的实验流程构建多重miRNA文库，并在Illumina HiSeq 2000测序平台上完成测序。测序数据前期处理阶段，使用Illumina CASAVA 1.8.2软件完成图像分析、碱基识别、解复用以及BCL格式文件到FASTQ格式文件的转换；再通过mirExpress软件去除接头序列，并对FASTQ序列进行3'端接头剪切。将处理后的序列比对至hg19（GRCh37）版本的人类参考基因组，比对工具为STAR（v.2.5.2a），且比对过程中不允许存在参考序列错配。最后使用HTSeq-count（HTSeq v 0.6.1p1 Python工具包）的‘intersection-strict’模式对测序读段进行计数，依据miRBase v.20数据库统计成熟miRNA的表达丰度。