title: NextSeq 550 sequencing; Induction of type I interferons in macrophages requires MEF2A and is targeted by prostaglandin E2
design: Induction of type I interferons in macrophages requires MEF2A and is targeted by prostaglandin E2
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 10^4 – 5 x 10^6 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, and were stimulated as described. BMDMs or iMACs were stimulated for 2 hours at indicated condition 50,000 cells were collected and centrifuged at 450xg for 5 minutes. Then, the transposition reaction was performed using digitonin 1%, Tn5 transposase and TD Buffer for 45 minutes at 37°C. Immediately following transposition, the reaction was stopped using a solution of 900 mM NaCl and 300 mM EDTA, 5% SDS and Proteinase K for 30 minutes at 40°C.  Transposed DNA fragments were purified using AMPure XP beads, barcoded with Illumina Nextera dual indexes and PCR amplified with KAPA HiFi PCR Kit. Then, the concentration of the library was determined using Qubit 3.0 and size distribution was assessed using Agilent 4200 TapeStation system.