Illumina HiSeq 2000 paired end sequencing; RNA-Seq of human primary monocytes treated with inflammatory and anti-inflammatory stimuli at different and sequential time points
RNA-Seq of human primary monocytes treated with inflammatory and anti-inflammatory stimuli at different and sequential time points
Anti-Citrullinated Protein Antibody-containing Immune Complexes (ACPA-IC) refers to 25 g/ml of antibody plus 6 µg/ml of antigen incubated for 1h at 37°C; Ab and Ag were isolated from serum of patients affected by Rheumatoid Arthritis. LPS was derived from E. coli serotype O55:B5, PGN-BS was derived from B. subtilis, . Cells were harvested in 700 µl of Qiazol (Qiagen, Hilden, Germany) at 0, 2, 4, 14, 24, 48, 72, and 96 h. Total RNA was extracted from monocytes of 6 individual donors (3 for the “resolving model”: 0, 4-48 h; and 3 for the “persistent model”: 0, 4-96 h), using Qiagen miRNeasy kit (Qiagen), quantified spectrophotometrically (ND-1000; NanoDrop Technologies, Wilmington, DE), and checked for integrity by microcapillary electrophoresis (Agilent 2100 Bioanalyzer; Agilent Technologies, Palo Alto, CA). According to the TruSeq RNA Sample Prep Kit v2 (Illumina). mRNA isolation and fragmentation: 200ng total RNA sample was purified by oligo-dT beads, then poly (A)-containing mRNA were fragmented into small pieces with Elute, Prime, Fragment Mix . cDNA synthesis: First-strand cDNA was generated by First Strand Master Mix and Super Script II(Invitrogen) reverse transcription(Reaction condition:25°C for 10 min;42°C for 50 min;70°Cfor 15 min). Then adding Second Strand Master Mix to synthes the second-strand cDNA (16°C1h). End repair, add A and adaptor ligation: the purified Fragmented cDNA combine with End Repair Mix, incubate at30°C 30min. Purify the end-repaired DNA with Ampure XP Beads (AGENCOURT). Then add A-Tailing Mix , mix well by pipetting, incubate at 37°C 30 min. Adapter Ligation : Combine the Adenylate 3'Ends DNA, RNA Index Adapter and Ligation Mix, mix well by pipetting. Incubate the ligate reaction at 30°C 10 min. Purify the end-repaired DNA with Ampure XP Beads (AGENCOURT). RCR: Several rounds of PCR amplification with PCR Primer Cocktail and PCR Master Mix were performed to enrich the cDNA fragments. Then the PCR products are purified with Ampure XP Beads (AGENCOURT).