Qubit cdna hs assay kits

RNA isolation and cDNA preparation of FACS-sorted fibroblasts were performed using the SMARTer^® Universal Low Input RNA Kit (Takara Bio, Saint-Germain-en-Laye, France) according to the manufacturer’s instructions. Prepared cDNA was purified by immobilization on AMPure XP beads (Beckman Coulter), and quantified using Qubit™ cDNA HS Assay Kits (ThermoFisher Scientific, Dreieich, Germany). Quality of purified cDNA was checked using the Agilent 2100 Bioanalyzer^® with High Sensitivity DNA Chips. Purified cDNA of sufficient quality was sheared using a M220 focused ultrasonicator (Covaris, Brighton, UK), yielding cDNA fragments around 400 bp. Fragmented cDNA was then used to prepare libraries using the SMARTer ThruPLEX DNA-Seq Kit (Takara Bio) according to the manufacturer’s instructions. Amplified libraries were purified by immobilization on AMPure XP beads, and quality and DNA content were checked using High Sensitivity DNA Chips as well as again with Qubit™ cDNA HS Assay Kits. Libraries were diluted, denatured according to Illumina Denture and Dilute Libraries Guide, and mixed with PhiX control (8%). Six to eight libraries were loaded on one sequencing cartridge of the TG NextSeq^® 500/550 High Output Kit v2 (75 cycles) (Illumina, Eindhoven, The Netherlands) and sequencing was performed on the NextSeq platform (Illumina).

Total RNA of non-treated and efferocytotic Mφ (6 biological replicates each) was isolated using RNeasy Micro Kit (Qiagen) according to the manufacturer’s protocol. cDNA library preparation was carried out using QuantSeq 3’ mRNA-Seq Library Prep Kit FWD from Illumina (Lexogen) according to the manufacturer’s procedure. RNA and DNA quantification was done using Qubit cDNA HS Assay Kits (ThermoFisher Scientific) and quality control was performed using an Agilent 2100 Bioanalyzer with RNA Nano Chip (Agilent) as well as High Sensivity DNA chips (Agilent). Libraries were diluted and denatured according to the Illumina Denature and Dilute Libraries Guide, followed by mixing with 1% Phix Control (Illumina). 12 Libraries were loaded on one sequencing cartridge of the TG NextSeq 500/550 High Output Kit v2 (75 cycles) (Illumina) and RNA sequencing was performed on a NextSeq500 system (Illumina).