Next generation expression analysis of wild type and Acaca-/- murine small intestinal organoids

Purpose: The goal of this study is to compare NGS-derived transcriptomes of wild type mouse small interstinal organoids and organoids deficient for the enzyme acetyl-coa-carboxylase (ACC) 1 (Acaca). Methods: mRNA profiles were generated from organoids at 24h and 96h upon in vitro deletion of the Acaca gene and the respective non-deleted wild type controls in triplicates. Total RNA was isolated using RNeasy Micro Kit (Qiagen) and libraries were prepared using NEBNext Single Cell/Low Input RNA Library Prep (New England BioLabs). The libraries were sequenced on Illumina NovaSeq 6000 using NovaSeq 6000 S2 Reagent Kit (100 cycles, paired end run). Each FASTQ file gets a quality report generated by FASTQC tool. Before alignment to reference genome each sequence in the raw FASTQ files were trimmed on base call quality and sequencing adapter contamination using Trim Galore! wrapper tool. Reads shorter than 20 bp were removed from FASTQ file. Trimmed reads were aligned to the reference genome (GRCm38.) using open source short read aligner STAR (https://code.google.com/p/rna-star/) with settings according to log file. Results: the sequencing depth of our libraries is > 3 x107 reads per RNA sample. PCA analysis revealed a close relationship between WT and ACC1-deficient organoids at 24h, whereas samples at 96h were distinct from each other. Lack of ACC1 strongly reduced the expression of genes associated with intestinal epitelial stem cells. Moreover, GSEA revealed downregulation of pathways associated with DNA replication, cell cycle and chromosome segregation in ACC1-deficient organoids. Conclusions: Our study highlights the importance of ACC1-mediated cellular fatty acid synthesis for the maintenance of intestinal epithelial stem cells in mouse intestinal oganoids. mRNA profiles of wild type and ACC1-deficient murine small intestinal organoids

一、研究目的：本研究旨在对比野生型小鼠小肠类器官与乙酰辅酶A羧化酶1（acetyl-coa-carboxylase 1，ACC1，基因名Acaca）缺陷型小鼠小肠类器官的下一代测序（Next-Generation Sequencing，NGS）转录组。
二、研究方法：在体外敲除Acaca基因后的24小时与96小时，分别从类器官中提取样本，同时设置未敲除的野生型对照组，每组均设置三次生物学重复，以此生成mRNA表达谱。总RNA提取采用RNeasy Micro试剂盒（Qiagen公司），文库构建使用NEBNext单细胞/低起始量RNA文库制备试剂盒（New England BioLabs公司）。文库在Illumina NovaSeq 6000测序平台上，采用NovaSeq 6000 S2试剂套件（100个循环，双端测序模式）完成测序。每条FASTQ文件均通过FASTQC工具生成质量评估报告。在将序列比对至参考基因组前，使用Trim Galore!封装工具对原始FASTQ文件中的序列进行碱基质量修剪与测序接头污染去除，并移除长度短于20 bp的读段。将修剪后的读段比对至参考基因组（GRCm38），采用开源短读段比对工具STAR（https://code.google.com/p/rna-star/），比对参数参照对应日志文件设置。
三、研究结果：本研究中每个RNA样本的测序深度均超过3×10^7条读段。主成分分析（Principal Component Analysis，PCA）结果显示，野生型与ACC1缺陷型类器官在24小时时聚类特征相近，而96小时的样本则呈现显著分离。ACC1缺失会显著下调与小肠上皮干细胞相关的基因表达。此外，基因集富集分析（Gene Set Enrichment Analysis，GSEA）结果显示，ACC1缺陷型类器官中与DNA复制、细胞周期及染色体分离相关的通路均出现下调。
四、研究结论：本研究证实了ACC1介导的细胞脂肪酸合成对维持小鼠小肠类器官内小肠上皮干细胞稳态的重要性。本数据集包含野生型与ACC1缺陷型小鼠小肠类器官的mRNA表达谱。