title: Illumina NovaSeq 6000 sequencing; RNAseq of primary human monocyte derived macrophages (MDM) and differentiated THP-1 macrophages exposed to graphene-related (nano)materials (GRMs)
design: RNAseq of primary human monocyte derived macrophages (MDM) and differentiated THP-1 macrophages exposed to graphene-related (nano)materials (GRMs)
library_construction_protocol: Human THP-1 monocytes were obtained from the European collection of cell cultures (Lot number13 C011, ECOCC). Following thawing, cells were grown in suspension in 75 cm2 cell culture flasks, sub-cultured at least three times prior to experiments and grown in complete cell culture medium (RPMI-1640 medium) supplemented with 10% fetal calf serum (FCS), 1 % L-glutamine and 1% penicillin–streptomycin–neomycin. Cells were maintained at 37 °C and 5% CO2 in a humidified atmosphere and routinely sub-cultured twice a week at 70–80% confluency. For experiments, THP-1 monocytes were differentiated to macrophages with 200 nM phorbol 12-myristate 13-acetate (PMA) for 72 h before GRM exposure. THP-1 cells were seeded in 12 well plates with densities of 5 × 105 cells per well (3.9 cm2 growth area) in 1000 µl complete cell culture medium and cultivated for 72 h in the presence of 200 nM PMA. After differentiation, PMA containing medium was removed and the cells were washed with prewarmed phosphate buffered saline (PBS) twice before experiments. Peripheral blood was obtained from 7 donations from 4 donors under informed consent according to approval from the local cantonal ethics committee, St. Gallen, Switzerland (BASEC Nr. PB_2016-00816). Monocyte derived macrophages (MDM) were isolated from blood provided by health donors as described by Sallusto et al. with the modification of using CD14 magnetic beads (MicroBeads, Miltenyi Biotec) for monocyte isolation. The primary monocytes were seeded in 6 well plates with densities of 1× 106 cells per well in 2000 µl complete cell culture medium and were differentiated into MDM with 10 ng/ml macrophage colony stimulating factor (M-CSF, ) in complete cell culture medium  for 6 days. After differentiation cells were washed with prewarmed PBS twice before experiments. For all experiments, stock dispersions of the tested materials of 1 mg/ml in ultrapure water (GO) or sterile filtered (0.22 µm pores) 160 ppm Pluronic F-127 in ultra-pure water (GNP) were prepared by sonication for 10 min (ultrasonic bath, Sonorex Super RK 156 BH, Bandelin) prior to usage and used for maximum one month. Stock dispersions were diluted to the final experimental concentrations in complete cell culture medium, with a percentage of 10% GRM solution in medium and suspensions were gently pipetted on top of the cells growing in well plates. DQ was freshly dispersed in H2O, vortexed and applied with a final concentration of 100 µg/ml in complete cell culture medium. Total RNA was extracted from differentiated macrophages, which have been exposed to 5 or 20 μg/ml GRM, 100 µg/ml DQ, or complete cell culture medium with 10% H2O as the solvent control for  6h or 24 h, using miRNeasy® Mini (QIAGEN) according to the manufacturer's pro-tocol. DNase digestion has been performed either by RNase Free DNase Set (QIAGEN) to dis-card residual DNA contamination. The RNA samples were carefully labelled and stored at -80°C prior to sequencing. The quality of the RNA was determined with a Fragment Analyzer standard sensitivity RNA measurement (SS RNA kit (15 nt), Agilent, Waldbronn, Germany). The meas-ured concentrations (> 200 ng/µl) and RIN (>8) values qualified for a Poly-A enrich-ment strategy in order to generate the sequencing libraries applying the TruSeq mRNA Stranded Library Prep Kit (Illumina, Inc, California, USA). After Poly-A selection using Oligo-dT beads the mRNA was reverse-transcribed into cDNA. The cDNA was fragmented, end-repaired and poly-adenylated before ligation of TruSeq UD Indices (IDT, Coralville, Iowa, USA). The quality and quantity of the amplified sequencing libraries were validated using a Fragment Analyzer SS NGS Fragment Kit (1–6000 bp) (Agilent, Waldbronn, Germany).