title: Illumina NovaSeq 6000 sequencing; Genomic features enabling control of inflammatory gene expression by antagonistic signals
design: Genomic features enabling control of inflammatory gene expression by antagonistic signals
library_construction_protocol: Bone marrow cells were collected from femurs and tibias in 50 mL PBS, filtered through a 70 µm cell strainer and centrifuged 450 x g for 5 minutes. Red blood cells were lysed using 0.2% NaCl solution, followed by 1.6% NaCl solution. Cells were filtered through a 70 µm cell strainer and centrifuged 450 x g for 5 minutes. 5 x 104 – 5 x 106 bone marrow cells were plated and cultured in Iscove's Modified Dulbecco's Medium (IMDM) supplemented with 10% Fetal Bovine Serum (FBS), 20% L929-conditioned medium containing M-CSF, antibiotics (penicillin G 100 U/ml and streptomycin sulfate 100 U/ml), 2 mM L-glutamine and 5 µM 2-mercaptoethanol. After six days of culture, adherent cells were >99% Cd11b+ F4/80+, as assessed by flow cytometry BMDMs or iMACs were stimulated for four hours with the indicated stimuli Total RNA was extracted using ReliaPrep RNA Cell Miniprep System and quantified using NanoDrop 8000. RNA-Seq libraries were generated using the Smart-seq2 method (Picelli et al., 2014). Five ng of RNA were retrotranscribed, cDNA was PCR-amplified (15 cycles) and purified with AMPure XP beads. After purification, the concentration was determined using Qubit 3.0 and size distribution was assessed using Agilent 4200 TapeStation system. Then, the tagmentation reaction was performed starting from 0.5 ng of cDNA for 30 minutes at 55°C and the enrichment PCR was carried out using 12 cycles. Libraries were then purified with AMPure XP beads, quantified using Qubit 3.0 and sequenced.