Illumina NovaSeq 6000 paired end sequencing; RNA-seq of lung myeloid cells from WT and Ptgs2-cKO mice
RNA-seq of lung myeloid cells from WT and Ptgs2-cKO mice
KO-CD11bDC-2_p
WT and Ptgs2-cKO naïve mice were euthanized and lungs were harvested and digested. Lung single cell suspensions were prepared and CD11b+ DCs, CD103+ DCs and monocytes were sorted for RNA-seq. Cells were lysed and homogenized in TRIzol Reagent, then RNA was isolated using the miRNeasy Micro kit, according to manufacturers' protocols, including the optional DNase digest step. RNA concentration and quality were assessed using the Date Extracted (initials) spectrophotometer and the Nanodrop 2000 Assay.  Libraries were constructed using the SMARTer Stranded Total RNA-Seq Kit v2-Pico, according to the manufacturer's protocol.  Briefly, the protocol entails RNA fragmentation and cDNA synthesis, addition of Illumina-specific adapters containing a unique barcode sequence for each library, cleavage of ribosomal cDNA with Takara's ZapR v2 and R-Probes v2, and PCR amplification of uncleaved fragments.  The quality and concentration of the libraries were assessed using the D5000 ScreenTape and Qubit dsDNA HS Assay, respectively, according to the manufacturers' instructions.