Illumina NovaSeq 6000 paired end sequencing; Pinolenic acid exhibits Anti-inflammatory and anti-atherogenic effects in Peripheral blood-derived Monocytes from patients with Rheumatoid Arthritis
Rheumatoid arthritis (RA) Patients recruitment and PBMCs isolation For this study, 8 ACPA positive RA patients with active disease (DAS28 score range: 4.8-5.7); (mean age=57 years old) and they were on biologic anti rheumatic drugs. All patients were recruited from the Rheumatology Department at the University Hospital of Wales, signed informed consent was obtained from all participants. The study was approved by the Research Ethics Committee for Wales 3 (reference no. 12/WA/0045) University Hospital of Wales. Detailed demographic and laboratory data are described in (Supplementary Tables S1, S2) for intracellular cytokines and WGT assessment, respectively. PBMCs were isolated by standard Ficoll density gradient centrifugation and methodological details are provided in (Supplementary Data S1). All methods were performed in accordance with the relevant guidelines and regulations.Negative selection and sorting of purified monocytes To enrich purified monocytes for assessment of intracellular cytokines using flow cytometry, monocytes were recovered by negative selection in cascades by magnetic-activated cell sorting (MACS) pan monocyte isolation kit (Miltenyi Biotec, UK) following the manufacturer's protocol. Cells at a minimum density of 106 cells/ml were cultured in complete RPMI medium at 37°C in 5% CO2 controlled environment; the procedure was continued for cell surface/intracellular staining as described below or for the transcriptome analysis. For the transcriptome study, the magnetically purified monocytes were passed through a second step of purification using FACS Aria III based cell sorting. In brief, cells were prepared for sorting, enriched monocyte cell suspensions were incubated with 2 μg/ml Fc human serum immunoglobulin (BD Biosciences; UK) at 4°C for 10 min to reduce non-specific binding by Fc receptors, and a small volume was aliquoted and labelled as unstained that was used for compensation. The cells were then stained with live/dead™ (L/D) fixable viability stain (ThermoFisher Scientific, UK) with fluorochrome-conjugated antibodies to CD14 (BioLegend, USA) at a final dilution of 1/100. All experiments were controlled with appropriate isotype antibodies, compensation beads and unstained cells. To verify the purity and quantity of the sorted monocytes, purity check was performed under sterile conditions at 0-4°C. The purity of the monocytes was 83.3%, 83%, 92.3%, 85.2%, 89.2%, 87%, 90.7%, and 96% respectively for patients RA1- RA8. Sorted monocytes were washed with fresh RPMI medium, counted, and seeded in 6-well plates at a concentration of approximately 1x106 cells/mL in RPMI 1640 complete medium along with either 25 M PNLA or DMSO vehicle control for 24 hours before stimulation with LPS (100 ng/ml) or vehicle control for 4 hours. The cell suspensions were then collected and washed with Ca2+ or Mg2+ free Dulbecco's PBS medium (Thermo Fisher Scientific, UK). The adherent cells were detached from the plastic by addition of 1 ml accuatase cell dissociation reagent (Thermo Fisher Scientific, UK) pH 6.8/well, incubated for 5-8 min at 37°C with 5% CO2, and then washed. The cell suspension was centrifuged at 400 x g at room temperature for 5 min and kept at -80°C in 350 l buffer RLT (provided in RNeasy Mini kit) supplemented with 10% of 2-mercaptoethanol (Gibco Life Science, UK) as a lysate till RNA extraction. Total RNA was isolated using a RNeasy mini kit (Qiagen, Hilden, Germany) from purified monocytes following 100 ng/ml LPS and 25 µM PNLA or vehicle treatment. RNA was purified using a RNeasy kit on-column with DNase I digestion (Qiagen) as described in Supplementary data S3. The cell lysates were stored at −80°C and then passed through a series of spin columns to first bind genomic DNA, then RNA and finally to elute high-quality RNA using the methodology by Takala et al in 2021(6). A quality control check for RNA was assessed as described in Supplementary data S3. Library preparation including ribosomal RNA depletion  In collaboration with the Wales Gene Park (Cardiff University), 5 ng of total RNA was depleted of ribosomal RNA using the NEBNext® rRNA Depletion Kit (Human/Mouse/Rat), (New England BioLabs, (UK) Ltd). Ribosomal RNA depletion of each sample was assessed using the Agilent 4200 TapeStation with hsRNA ScreenTape (Agilent Technologies, Inc, UK).  The sequencing libraries were prepared using the NEB® Ultra™ II Directional RNA Library Prep Kit for Illumina® (New England BioLabs, (UK) Ltd) protocol. The steps included RNA fragmentation and priming, 1st strand cDNA synthesis, 2nd strand cDNA synthesis, adenylation of 3' ends, adapter ligation (adapter diluted 1:199) and PCR amplification (16-cycles). Following the addition of the PCR enrichment master mix, a unique index primer was added to each sample. The standard fragmentation procedure of 15 min at 94°C for samples for intact RNA (>7) was reduced to 8 min at 94°C for partially degraded RNA with RIN values 2-6. Except for the replacement of RNAClean® XP beads and SPRI select beads by AMPure XP beads (Beckman Coulter®) the manufacturer's instructions were followed. The libraries were validated using Agilent 4200 TapeStation and high sensitivity DNA ScreenTapes (Agilent Technologies, Inc, UK) to ascertain the insert size, and the Qubit® (Thermo Fisher Technologies, UK) was used to perform the fluorometric quantitation.