Denature and dilute libraries guide

Equal molar amounts of 13 libraries in total were pooled for a sequencing run. Accordingly, each analyzed library constitutes 7.7% of overall flowcell capacity. The combined library pool was denatured with NaOH and was finally diluted to 1.5 pM according to the Denature and Dilute Libraries Guide (Illumina). 1.3 ml of denatured pool was loaded on an Illumina NextSeq 550 sequencer using a High Output Flowcell for 75bp single reads (#FC-404-2005; 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).