title: GSM7984573: inflamed_CCR1_rep4; Mus musculus; RNA-Seq
library_construction_protocol: Cell suspensions were stained for 20 minutes at 4°C with 100 μL of fixable viability stain (eBioscience) and washed in FACS buffer (PBS containing 2mM EDTA and 2% FBS). Next, cells were stained for 20min at 4°C with 50 μL of subset-specific antibody cocktails (Supplementary Table 1) and washed in FACS buffer. For flow cytometry analysis, stained cells were fixed for 20min at 4°C in 100 μL of Fixation Buffer (BioLegend) and analysed on a BD LSRFortessa flow cytometer (BD Biosciences). For monocyte isolation and transcriptomic analysis, stained cells (Supplementary Table 2) were analysed on a FACS Aria sorter (BD Biosciences) without previous fixation. Monocytes expressing either CCR1/2 or CCR2 only were sorted in RLT buffer (Qiagen) containing 10 μL/mL of b-mercaptoethanol and stored at -80oC for RNA extraction. RNA from sorted monocytes was isolated using the RNeasy® Micro Kit (Qiagen) as per manufacturer's instructions. Next, mRNA libraries were prepared using the NEBNext® Single Cell/Low Input RNA Library Prep Kit for Illumina® (New England Biolabs). Paired-end sequencing was performed in a NextSeq2000 sequencing platform (Illumina) aiming for 40 million reads sequencing depth.