LC-MS and gene expression.xlsx

Metabolites of the fecal supernatants were analyzed at Chalmers Mass Spectrometry Infrastructure (Gothenburg, Sweden) using a non‐targeted liquid chromatography–mass spectrometry (LC‐MS) approach. Briefly, the analyses were carried out using reversed‐phase chromatography and hydrophilic interaction chromatography with both positive and negative electrospray ionization. The samples of each study group were analyzed in separate batches, which included its own quality control samples. The analytical workflow named “notame”, described by Zheng et al, was used to pre‐process the acquired data and included drift correction within and between batches, data imputation was done using the <em>missForest</em> R package and clustering of features was done to remove weak and repeated features(24). Log10 transformation was applied prior between‐batch‐correction to reduce possible batch effects caused by the instrument <br> For gene expression data, a custom RT² PCR Array (Qiagen) was analyzed in a Quantstudio 12K Flex Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) using the RT² qPCR SYBR Green ROX MasterMix (Qiagen). The gene expression custom array comprised 96 genes (supplementary table 1), including 5 housekeeping (HK) genes (GAPDH, HPRT1, RPLP0, ACTB, B2M) and quality 4 controls (positive PCR control, reverse transcription control, genomic DNA control, negative template control). All samples passed the quality controls. Missing values and CT values &gt;38 were set to 38. Genes were excluded from the analysis if &gt;50% of the samples had missing or very high CT values (CT&gt;36). In total, 18 genes were excluded from the Caco-2 cell analysis, and 19 genes were excluded from the colonoid analysis. Normalized values are expressed as 2-(CT Target gene-mean CT of HK genes)

粪便上清液中的代谢物于查尔姆斯质谱平台（Chalmers Mass Spectrometry Infrastructure，位于瑞典哥德堡）采用非靶向液相色谱-质谱联用（liquid chromatography–mass spectrometry，LC-MS）分析方法进行检测。简言之，本次分析采用反相色谱和亲水相互作用色谱，并结合正、负电喷雾电离模式。各研究组的样本均在独立批次中进行分析，每个批次均包含对应质控样本。采用郑等人所报道的名为“notame”的分析流程对原始数据进行预处理，该流程涵盖批次内与批次间的漂移校正；使用R包<em>missForest</em>完成数据填充，并通过特征聚类剔除弱信号与重复特征[24]。为降低仪器引入的潜在批次效应，在批次校正前对数据进行了Log10转换。
对于基因表达数据，采用RT² qPCR SYBR Green ROX预混液（Qiagen），在Quantstudio 12K Flex实时荧光定量PCR系统（美国加利福尼亚州福斯特城应用生物系统公司）中对定制化RT² PCR Array（Qiagen）进行检测分析。本次定制化基因表达芯片共包含96个基因（详见补充表1），其中涵盖5个管家（HK）基因（GAPDH、HPRT1、RPLP0、ACTB、B2M）以及4种质控对照：阳性PCR对照、反转录对照、基因组DNA对照与阴性模板对照。所有样本均通过质控检测。缺失值与CT值大于38的位点均被赋值为38。若某基因在超过50%的样本中存在缺失值或CT值大于36，则将该基因从分析流程中剔除。总计有18个基因被排除在Caco-2细胞分析之外，19个基因被排除在结肠类器官分析之外。归一化值以2-(靶基因CT值-管家基因平均CT值)的形式表示。