Title: RNA-seq analysis of b0-HUDEP2 and HUDEP2 cells following erythroid differentiation

Purpose: The goal of this study is to compare the altered transcriptional program (RNA-seq) and cellular adaptation to stress caused by excess a-globin synthesis. Results: 100bp paired-end polyA-enriched RNA sequencing were performed with a depth of minimum of 20 million read counts per sample. The sequencing library was sequenced on the Illumina HiSeq2500 at Azenta. Raw data then went through read quality assessment by FastQC (version 0.11.5), mapping to the human genome (GRch38) by the STAR aligner and gene-level reads quantification using the Rsubread package. RNA-seq data analysis identified 1870 upregulated and 1549 down regulated genes on Day 0 (FDR <=0.05) and 1924 upregulated and 1960 down regulated genes on Day 8 (FDR <=0.05). MA plot analysis of differentially expressed genes uncovered an altered transcriptional program favouring ?-globin expression not solely dependent upon the HRI ATF4-BCL11A axis. Instead, we noted attenuated expression of several key ?-globin co-repressors, including SOX6, ZBTB7A, KLF1 and GATA1, which coincided with increased expression of co-activators such as CBFB/NY-FB and GATA2 following erythroid differentiation. Gene Set Enrichment Analysis (GSEA) software was used to determine significantly enriched gene sets. Downregulated DEGs in ß0 HUDEP 2 cells in pathways related to haeme metabolism, hypoxia, MTORC1, unfolded protein response and TNFA/NFkB signalling were enriched. Meanwhile, upregulated DEGs in ß0 HUDEP-2 cells related to G-protein-coupled receptors (GPCR) signalling, IL-2/STAT5 and hypoxia signalling pathway were also retrieved. Pathways shown to be significantly altered in both ß0 HUDEP-2 cells and patient group include apoptosis, MAPK signalling and NFKB pathway thus provided complementary insights in transcriptome profiling. Conclusions: Our study represents analysis of HUDEP2 transcriptomes, with biologic replicates, generated by RNA-seq technology. The data analysis reported here should provide a framework for comparative investigations of expression profiles. We conclude that RNA-seq based transcriptome characterization would expedite genetic network analyses and permit the dissection of the cellular adaptation to intrinsic stress caused by excess a-globin synthesis. Overall design: Erythroid mRNA profiles of b0-HUDEP2and HUDEP2 cells on day 0 and day 8 differentiation.

研究目的：本研究旨在对比过量α-珠蛋白合成引发的应激状态下，经RNA测序（RNA-seq）检测到的转录调控程序改变与细胞适应性反应。
结果：本研究对每个样本完成了至少2000万读长计数的100bp双端polyA富集RNA测序，测序文库在Azenta的Illumina HiSeq2500平台上完成测序。原始数据首先通过FastQC（版本0.11.5）进行读段质量评估，随后通过STAR比对软件将测序读段比对至人类基因组GRch38，并使用Rsubread软件包完成基因水平的读段定量。
RNA-seq数据分析显示，在分化第0天，共鉴定出1870个上调基因与1549个下调基因（错误发现率FDR≤0.05）；在分化第8天，共鉴定出1924个上调基因与1960个下调基因（FDR≤0.05）。对差异表达基因的MA图分析表明，偏向β-珠蛋白表达的转录调控程序改变并非仅依赖HRI-ATF4-BCL11A信号轴。研究同时观察到多个关键β-珠蛋白共阻遏因子的表达水平减弱，包括SOX6、ZBTB7A、KLF1与GATA1；这一现象与红细胞分化后CBFB/NY-FB、GATA2等共激活因子的表达上调相契合。
本研究使用基因集富集分析（Gene Set Enrichment Analysis, GSEA）软件鉴定显著富集的基因集。在β0 HUDEP-2细胞中，下调的差异表达基因富集于血红素代谢、缺氧、MTORC1、未折叠蛋白反应以及TNFA/NFκB信号通路等相关通路。与此同时，该细胞中上调的差异表达基因则富集于G蛋白偶联受体（G-protein-coupled receptors, GPCR）信号、IL-2/STAT5与缺氧信号通路等。在β0 HUDEP-2细胞与患者组中均发生显著改变的通路包括细胞凋亡、MAPK信号通路与NFκB通路，这为转录组分析提供了互补的研究视角。
结论：本研究对HUDEP2细胞的转录组完成了分析，设置了生物学重复，测序数据由RNA-seq技术生成。本文报道的数据分析可为表达谱的比较研究提供标准化分析框架。本研究结论如下：基于RNA-seq的转录组表征可加速遗传网络分析，并助力解析过量α-珠蛋白合成引发的内在应激下的细胞适应性机制。
总体设计：本研究的总体实验设计为：对分化第0天与第8天的β0-HUDEP2细胞及HUDEP2细胞进行红细胞系mRNA表达谱分析。