design: Rheumatoid Synovial Subsets of CD3+ T cells defined by PD-1+ and PD-1- Cells Drive Fibroblasts into Distinct Endotypes
library_construction_protocol: Fibroblasts from HC skin in passage 3 were thawed and cultured in a T175 flask (Sarstedt, cat. no. 83.3911.002) in DMEM low glucose media (VWR, cat. no. 392-0407). When confluency was reached cells were trypsinized and seeded into a 96-well plate and stimulated by adding 20% supernatant from PD-1+/- cultures (n=3), IFNγ (10 µg/ml) (n=3), PD-1+/- T cells (n=3), or no stimulation (n=3). After 24 hours T cells were removed. RNA was purified from the co-cultured fibroblasts after washing with PBS and cell lysis. RNA-purification was done using the RNeasy Mini Kit (Qiagen, cat. no. 74104) (n=1 in triplicates), and the Genomic DNA was removed by using the RNase-Free DNase Set (Qiagen, cat. no. 79254).  The RNA integrity number and concentration were measured on Bioanalyzer RNA Nano chip (Agilent, cat.no. 5067-1511). The SMARTer Stranded Total RNA Sample Prep Kit - HI Mammalian (Takara, Japan, cat.no. 634876) was used for library preparation with 200ng of the purified RNA as input following the manufacturer`s protocol. The Bioanalyzer High sensitivity DNA analysis kit (Agilent, cat.no. 5067-4626) was used to determine the size of the library fragments and the Qubit dsDNA High Sensitivity Kit (Invitrogen, cat. no. Q32851) was used to quantify the library.