GSM2772497: H-398_LPS; Homo sapiens; RNA-Seq
Monocytes were sorted from frozen PBMC on a BD Influx cell sorter (BD Biosciences). After sorting, cells were lysed ex vivo or resuspended in complete RPMI with 10% FCS at 1x106 cells/mL and stimulated in 96-well flat-bottom plates for 6 or 24h ± LPS. Monocyte viability, assessed by FACS, decreased slightly with culture time and stimulation: median viability (IQR) ex vivo, at 6h medium, 6h LPS, 24h medium and 24h LPS was 98.3% 482 (1.7), 97% (2.2), 93.9% (5.2), 91.7% (4.5) and 90.7% (12.3), respectively. There were no differences in viability between healthy and sJIA monocytes. After incubation, supernatants were collected and cells were lysed with RLT buffer supplemented with 1% β-mercaptoethanol (Qiagen, Valencia, CA, USA) for RNA-Seq analysis. RNA from cell suspensions was purified using the RNAqueous-Micro Total RNA Isolation Kit (Thermo Fisher Scientific). RNA integrity was assessed using RNA 6000 Pico or RNA 6000 Nano assay on Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). RNA concentration and purity were assessed using the NanoDrop 8000 (Thermo Fisher Scientific). All procedures were performed according to the manufacturer’s instructions. Total RNA was converted to cDNA using 20 ng of RNA as input for the Ovation RNA-Seq System V2, and barcoded Illumina library constructs were produced using Ovation Ultralow Library System V2 (NuGEN Technologies, San Carlos, CA, USA) following the manufacturer’s instructions. Library fragment size and molar concentration were determined using Agilent High Sensitivity DNA Chip on 2100 Bioanalyzer, and qPCR data obtained by KAPA BioSystems Library Quantification qPCR Kit for Illumina Platform (Kapa Biosystems, Wilmington, MA, USA) on Viia7 Real-Time PCR System (Thermo Fisher Scientific). RNA-Seq libraries were sequenced on the Illumina HiSeq 2500 sequencing system, using SBS v3 chemistry and two high-output flow cells targeting 70-80 million 2X75 paired-end reads per library.