title: Illumina NovaSeq 6000 paired end sequencing; Sequencing of synovial tissue fibroblasts from Rheumatoid Arthritis patients that were co-cultured with inflammatory macrophages.
design: Sequencing of synovial tissue fibroblasts from Rheumatoid Arthritis patients that were co-cultured with inflammatory macrophages.
library_construction_protocol: To obtain macrophages, CD14pos monocytes isolated from healthy donors (n=6) were plated in a 24-well plate in 3 ml complete medium (RPMI with 10% FCS, Penicillin/Streptomycin 100/U/ml and L-Glutamine 2mM) containing M-CSF (PeproTech) at the concentration of 50 ng/ml  On day 3, macrophages were pre-treated with LPS (1 ng/ml) for 4h. For the last 2h, MerTK specific inhibitor, UNC106238 (Aobious) was added to a half of the wells at the concentration 250 uM. Cells were then washed with PBS and transwell inserts (0.4 um pore size) containing 3x105 Rheumatoid Arthritis FLS derived from 4 different patients (n=4) were added to the wells to generate a co-culture system. After 48h, FLS growing on the transwell inserts were moved into new 24 wells and lysed with RLT buffer with 1% beta-mercaptoethanol (Qiagen) and stored at -80oC till the RNA isolation step.  We have 6 separate experiments with FLS from 4 different RA patients and macrophages from 6 different healthy donors.  2 FLS samples were used twice to react with distinct macrophage donors.   FLS co-cultured with macrophages pre-treated with LPS and MerTK inhibitor are named FLS + Mac (LPS+MerTK inhibitor)  RNA was isolated using RNEasy micro-kit (Qiagen). The samples containing FLS co-cultured with macrophages pre-stimulated with LPS are named FLS+Mac (LPS) while FLS co-cultured with macrophages pre-treated with LPS and MerTK inhibitor are named FLS + Mac (LPS+MerTK inhibitor). cDNA synthesis and amplification were performed by using SMART-seq v4 Ultra Low Input RNA Kit for Sequencing (cat. no. 634890, Takara) starting with 10 ng of total RNA, following the manufacturers protocol. 10 ng of amplified cDNAs were sheared prior to preparing the final libraries using the Bioruptor® Pico system (Diagenode, 24 cycles of 30 sec on and 30 sec off)
attributes.tag: Experimental Factor: compound
attributes.value: UNC106238
attributes.tag: Experimental Factor: dose
attributes.value: 250
attributes.tag: Experimental Factor: stimulus
attributes.value: lipopolysaccharide; 1 nanogram per milliliter
attributes.tag: Experimental Factor: specimen
attributes.value: 1534