RNAseq 数据

通过TRIzol试剂进行总RNA提取，并使用NanoDrop ND-1000分光度计评估其质量，确保RIN值大于7.0，并通过琼脂糖凝胶进行验证。使用 Dynabeads 从提取的总 RNA 中分离 Poly （A） RNA，然后使用 SuperScript™ II 进行片段化和 cDNA 合成。随后的 U 标记的第二链 DNA 创建涉及大肠杆菌 DNA 聚合酶 I 和 RNase H，以 A 碱基添加和接头连接加帽。在PCR扩增之前，用AMPureXP珠子进行大小选择。在Illumina Novaseq™ 6000平台上使用双端150 bp读数对文库进行测序。测序后，fastp 促进了初始数据清理，HISAT2 能够与 GRCh38 人类参考基因组进行比对。用StringTie和gffcompare进行转录组组装，在FPKM中定量mRNA表达水平。使用edgeR进行差异表达分析，应用>2或<0.5的倍数变化滤波器，显着性水平为p < 0.05。

Total RNA was extracted using TRIzol reagent, and its quality was assessed with a NanoDrop ND-1000 spectrophotometer to ensure a RIN value greater than 7.0, followed by verification via agarose gel electrophoresis. Poly(A) RNA was isolated from the extracted total RNA using Dynabeads, then fragmented and subjected to cDNA synthesis using SuperScript™ II. The subsequent generation of U-labeled second-strand DNA involved Escherichia coli DNA polymerase I and RNase H, with A-tailing and adapter ligation performed afterward. Size selection was carried out using AMPure XP beads prior to PCR amplification. The library was sequenced on the Illumina NovaSeq™ 6000 platform using paired-end 150 bp reads. After sequencing, initial data cleaning was performed using fastp, and alignment to the human reference genome GRCh38 was conducted via HISAT2. Transcriptome assembly was accomplished using StringTie and gffcompare, with mRNA expression levels quantified in units of FPKM. Differential expression analysis was performed using edgeR, with a fold change filter of >2 or <0.5 applied, and a significance threshold set at p < 0.05.