title: GSM9376926: D29_GA-AFUC monocytes; Mus musculus; RNA-Seq
library_construction_protocol: Three days after MA30 infection, mice were anesthetized with ketamine/xylazine (100 and 10 mg/kg via intraperitoneal injection, respectively), and 100 μL of CD45.2 antibody (APC; clone 104; Biolegend) was administered at a 1:10 dilution in PBS by retroorbital injection to identify intravascular cells. Two min later, mice were euthanized and then perfused with sterile PBS. Lungs were digested in 50 μL of Liberase Tm (5 mg/mL, Roche) and 12.5 μl of DNase I (10 mg/mL, Sigma-Aldrich) in 5 mL of HBSS at 37°C for 35 min. Single-cell suspensions were pre-incubated with Fc block antibody (BioLegend) in PBS + 2% heat-inactivated FBS + 1 mM EDTA for 20 min at 4°C. Cells were then divided into two groups for sorting. For interstitial macrophages, cells were stained with antibodies against CD45 (PE-Cy7; clone 30-F11; Biolegend), MHCII (BV421; clone M5; Biolegend), CD88 (PE; clone 20/70; Biolegend), and CX3CR1 (BV510; clone SA011F11; Biolegend). For classical monocytes, cells were stained with antibodies against CD45 (PE-Cy7; clone 30-F11; Biolegend), Ly6G (BV510; clone 1A8; Biolegend), Ly6C (PE; clone HK1.4; Biolegend), and CD115 (BV421; clone AFS98; Biolegend). All samples were stained with fixable viability dye (zombie green), True-Stain Monocyte Blocker (5 μL per sample), and Brilliant Stain Buffer Plus (10 μL per sample). All antibodies were used at a dilution of 1:200, with the exceptions of MHCII BV421, CD88 PE, and Ly6C, which were used at 1:100, and the viability dye, which was used at 1:2,000. Following incubation in PBS + 2% heat-inactivated FBS + 1 mM EDTA for 20 min at 4°C, cells were sorted on a MACSQuant Tyto instrument (Miltenyi Biotec). Following cell sorting, RNA was extracted using the Arcturus PicoPure RNA isolation kit (Thermo Fisher Scientific) according to the manufacturer's instructions. RNA purity was confirmed using a NanoPhotometer® spectrophotometer (IMPLEN). RNA concentration and quality were assessed using the RNA Nano 6000 Assay Kit of the Bioanalyzer 2100 system (Agilent Technologies). One µg of RNA per sample was used as input material for the RNA sample preparations. Sequencing libraries were generated using NEBNext® UltraTM RNA Library Prep Kit for Illumina® (NEB, USA) following the manufacturer's recommendations, and index codes were added to attribute sequences to each sample. Briefly, mRNA was purified from total RNA using poly-T oligo-attached magnetic beads. Fragmentation was carried out using divalent cations under elevated temperature in NEBNext First Strand Synthesis Reaction Buffer (5X). First strand cDNA was synthesized using a random hexamer primer and M-MuLV Reverse Transcriptase (RNase H-). Second strand cDNA synthesis was performed using DNA Polymerase I and RNase H. Remaining overhangs were converted into blunt ends via exonuclease/polymerase activities. After adenylation of 3' ends of DNA fragments, NEBNext Adaptor with hairpin loop structure were ligated to prepare for hybridization. To select cDNA fragments of 150~200 bp in length, the library fragments were purified with AMPure XP system (Beckman Coulter, Beverly, USA). Then, 3 µL of USER Enzyme (NEB) was added with size-selected, adaptor-ligated cDNA at 37°C for 15 min followed by 5 min at 95°C before PCR with Phusion High-Fidelity DNA polymerase, Universal PCR primers and an Index Primer. Finally, PCR products were purified (AMPure XP system), and the library quality was assessed on an Agilent Bioanalyzer 2100 system. The clustering of the index-coded samples was performed on a cBot Cluster Generation System using PE Cluster Kit cBot-HS (Illumina) according to the manufacturer's instructions. After cluster generation, the library preparations were sequenced on an Illumina platform, and paired-end reads were generated.