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+.