Differentially expressed genes detected with RNA sequencing analysis of osteoprogenitor populations in synovial joints of mice with antigen-induced arthritis

Antigen-induced arthritis (AIA) was induced in C57BL6 mice by immunization with methylated bovine serum albumin (mBSA) and subsequent intra-articular injection of mBSA. Non-immunized (NI) mice were injected with phosphate buffered saline at all timepoints. Ten days after intra-articular injection knee joints were harvested and synovial cells were released by collagenase type IV injection into joint cavitied, and fluorescence-activated cell sorting (FACS) was used to sort 200-500 TER119–CD31–CD45–CD51+CD200+CD105– cells from NI mice (NI 200+, Sample 1-4) and mice with AIA (AIA 200+, Sample 5-9) and, TER119–CD31–CD45–CD51+CD200–CD105+ cells from mice with AIA (AIA 105+, Sample 11-14) using BD FASCAria IIu. For each sample, 200-500 live (DAPI–) cells were sorted directly into cell lysis buffer from Smartseq v4 Ultra® Low Input RNA Kit for Sequencing (TakaRa, Kyoto, Japan). ERCC RNA Spike-In Mix (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA) were added to lysed cell samples. cDNA amplicons were created using SmartSeq v4 Ultra® Low Input RNA Kit for Sequencing (TakaRa) and libraries were prepared using Nextera XT DNA Library Preparation Kit (Illumina, San Diego, CA, USA). Libraries were sequenced using NextSeq 500 (Illumina) instrument and raw files are available at GSE148130. After quality control of raw reads, reads were trimmed, aligned, assembled and quantified using cutadapt (Martin, EMBnet Journal, 2011), HISAT2 (Kim, Nat Methods, 2015) and StringTie (Pertea, Nat Biotecnol, 2015), and normalized and filtered in egdeR package (Robinson, Bioinformatics, 2010). Limma voom (Law, Genome Biol, 2014) was used to detect differentially expressed genes. Genes with absolute fold change (FC) > 1.5 and p value (Benjamini-Hochberg) < 0,05 were considered differentially expressed and are listed in the tables, together with their FC, expressed as log2FC, and adjusted p value (Benjamini-Hochberg). The table provides Ensembl gene ID, Entrez gene ID, MGI gene symbol, gene biotype, chromosome name, start and end position of all differentially expressed genes. It also contains expression values, expressed as log2 counts per million mapped reads (CPM), for each sample. Table 1 contains comparison of NI 200+ and AIA 200+, Table 2 NI 200+ and AIA 105+ and Table 3 AIA 200+ and AIA 105+.

抗原诱导性关节炎（Antigen-induced arthritis, AIA）通过甲基化牛血清白蛋白（methylated bovine serum albumin, mBSA）免疫C57BL/6小鼠，随后向其关节腔内注射mBSA构建。未免疫（Non-immunized, NI）小鼠在所有时间点均注射磷酸盐缓冲液。关节腔注射后第10天，摘取膝关节，通过向关节腔注射IV型胶原酶分离滑膜细胞，随后使用BD FASCAria IIu流式细胞仪，通过荧光激活细胞分选（fluorescence-activated cell sorting, FACS）从NI小鼠（NI 200+，样本1-4）与AIA模型小鼠（AIA 200+，样本5-9）中分选200-500个TER119–CD31–CD45–CD51+CD200+CD105–细胞，同时从AIA模型小鼠（AIA 105+，样本11-14）中分选TER119–CD31–CD45–CD51+CD200–CD105+细胞。
对于每个样本，将200-500个活细胞（DAPI–）直接分选至Smartseq v4 Ultra® 超低投入量RNA测序试剂盒（Takara，日本京都）的细胞裂解液中。向裂解后的细胞样本加入ERCC RNA Spike-In Mix（Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA）。使用SmartSeq v4 Ultra® 超低投入量RNA测序试剂盒（Takara）制备cDNA扩增产物，再使用Nextera XT DNA文库制备试剂盒（Illumina, San Diego, CA, USA）构建测序文库。使用NextSeq 500（Illumina）测序仪对文库进行测序，原始数据已上传至GSE148130。
对原始测序读段进行质控后，使用cutadapt（Martin, 《EMBnet Journal》, 2011）、HISAT2（Kim, 《Nature Methods》, 2015）与StringTie（Pertee, 《Nature Biotechnology》, 2015）完成读段修剪、比对、组装与定量，并通过edgeR软件包（Robinson, 《Bioinformatics》, 2010）进行标准化与过滤。使用Limma voom（Law, 《Genome Biology》, 2014）检测差异表达基因。以绝对折叠变化（fold change, FC）>1.5且Benjamini-Hochberg校正后p值<0.05作为差异表达基因的筛选标准，相关基因及其log2转换后的FC值、校正后p值均列于表格中。
表格包含所有差异表达基因的Ensembl基因ID、Entrez基因ID、MGI基因符号、基因生物型、染色体名称及起始与终止位置，同时包含每个样本的表达值（以log2转换后的每百万比对读段计数CPM表示）。其中表1为NI 200+与AIA 200+的比较，表2为NI 200+与AIA 105+的比较，表3为AIA 200+与AIA 105+的比较。