Transcriptomic Profiling of Ly6G− IA/IE− CD45+ CD11b+ CD115+ Mouse Monocytes: Impact of In Vivo Platelet Depletion and LPS Challenge
Transcriptomic Profiling of Ly6G− IA/IE− CD45+ CD11b+ CD115+ Mouse Monocytes: Impact of In Vivo Platelet Depletion and LPS Challenge
In vivo, platelet depletion was performed in female C57BL/6J mice (12 weeks) by intravenous (i.v.) injection of 2 mg/kg of polyclonal rat anti-mouse GPIbα (R300) or same amounts of a polyclonal rat IgG none-immune (C301) antibody as control (Emfret Analytics). Mice were then challenged 12 hours later with i.v. injection of 2 mg/kg LPS or PBS as control. Blood was collected after 3 h, and PBMCs were isolated by density gradient centrifugation in Ficoll Paque PLUS. Mouse monocytes were separated from PBMCs by FACS-sorting gating on Ly6G−, IA/IE−, CD45+, CD11b+, and CD115+ cells and sorted directly into QIAzol lysis reagent. Lysates were frozen in liquid nitrogen and sent to GENEWIZ Azenta Life Sciences for bulk RNA sequencing.   For bulk RNA sequencing, samples were processed according to GENEWIZ (Azenta Life Sciences) using TRIzol extraction protocol. QC was done on QuBit and TapeStation.   RNA extraction was performed, followed by a library preparation (Azenta, GeneWiz).  SmartSeq HT library preparation kit with maximum input volume as the RNA amount was below QuBit detection limit.  mRNA sequencing via polyA selection: 1) mRNA Enrichment, mRNA Fragmentation, and Randon Priming; 2) First and second strand cDNA synthesis; 3) End Repair, 5' Phosphorylation, and dA-Tailing; 4) Adaptor Ligation, PCR Enrihment, and Sequencing.