DNBSEQ-T7 paired end sequencing; RNA-Seq of rheumatoid arthritis synovial fibroblasts treated with CDK4/6 inhibitor and TNF+IL-1b stimulation
RNA-Seq of rheumatoid arthritis synovial fibroblasts treated with CDK4/6 inhibitor and TNF+IL-1b stimulation
Cells were lysed with RLT buffer RASFs were obtained from 5 individual patients and were maintained in the DMEM with 10%FBS and 1% penicillin and streptomycin. Synovial tissues were obtained from patients with RA when they underwent joint replacement surgery or synovectomy. All the patients fulfilled the criteria of the American College of Rheumatology and consent forms were completed by the patients before surgery. RASFs were isolated and cultured as described elsewhere. The experiments used proliferating RASFs (from passage 3 or 4). RASFs were plated to 10cm dish and incubated overnight. Next day, RASFs were treated with 2uM of palbociclib or DMSO. After 24 hours, RASFs were treated with 0.2ng/ml of TNF+IL-1b respectively for 24 hours. RASFs were lysed with RLT buffer RNeasy Plus Mini Kit (QIAGEN) according to the manufacture's protocol 1)mRNA enrichment and purification: Oligo dT Selection to enrich the mRNA (For total RNA extracted from human whole blood, globin mRNA are depleted ); 2)RNA fragmentation and cDNA synthesis (second-strand cDNA synthesis with dUTP instead of dTTP); 3)End repair, add A and adaptor ligation; 4)PCR; 5)Circularization and make DNB; 6) Sequencing on DNBSEQ platform