Remodeling of cell-type specific transcriptomes upon T cell stimulation in Systemic lupus erythematosus versus Sjögren's syndrome
Remodeling of cell-type specific transcriptomes upon T cell stimulation in Systemic lupus erythematosus versus Sjögren's syndrome
Lithium Heparin (LiHep)-anticoagulated blood was withdrawn from a total of 24 healthy donors, 25 SLE patients and 23 pSS patients. PBMCs were isolated from LiHep-anticoagulated blood. The collected mononuclear cells were washed twice with PBS. cells were rested for 30 min at 37°C in ImmunoCult-XF T cell expansion medium (Stemcell). 96-well plate were coated with 0.1 µg/ml anti-CD3 (OKT3, eBioscience) for 2h at 37°C or PBS as control, washed two times with PBS, blocked with PBS/1% BSA for 30 min and washed two times with PBS. 1x106 PBMCs were seeded per well and incubated at 37°C and 5% CO2 in a humidified incubator for 18hours. The PicoPure RNA Kit (Thermo Fisher) was used for purification of total RNA from cells. Library generation has been completed using the SMARTer cDNA Synthesis and Nextera XT chemistry. With 500pg total RNA, cDNA pools were prepared using the SMART-Seq v4 Kit (Clontech Laboratories) to generate full-length cDNA from total RNA.