NextSeq 550 paired end sequencing; Induction of type I interferons in macrophages requires MEF2A and is targeted by prostaglandin E2
Induction of type I interferons in macrophages requires MEF2A and is targeted by prostaglandin E2
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.  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, and were stimulated as described.  BMDMs were stimulated for 4 hours, as indicated. scRNA-Seq libraries were generated using a microfluidics-based approach on Chromium Single-Cell Controller (10X Genomics) using the Chromium Single Cell 3′ Reagent Kit v2 according to the manufacturers' instructions. Briefly, single cells were partitioned in Gel Beads in Emulsion (GEMs) and lysed, followed by RNA barcoding, reverse transcription and PCR amplification (12 cycles). The concentration of the scRNA-seq libraries was determined using Qubit 3.0 and size distribution was assessed using an Agilent 4200 TapeStation system.