title: GSM6205061: Heart Macrophages, LOY BMT, bulkRNAseq [41x1]; Mus musculus; RNA-Seq
library_construction_protocol: For bulk RNA-Seq analysis of the peripheral blood neutrophils, whole blood was collected and incubated with RBC lysis buffer. After staining with fluorescent-labeled monoclonal antibodies, a total of 10,000 blood neutrophils (Ly6G+CD115-RFP+) were sorted directly into 350 ul of buffer RLT (Qiagen, Cat. #79216) containing 1% b-mercaptoethanol using Influx Cell Sorter platform with 100 mm nozzle and flow pressure set to 20 psi (BD Biosciences). For cardiac macrophages and neutrophils, hearts were flushed with 15 ml of cold PBS from apex and excised. Right ventricles were removed, and left ventricles were minced and digested in collagenase I (450 U/ml), collagenase XI (125 U/ml), hyaluronidase (450 U/ml), and DNase I (60 U/ml) (Sigma-Aldrich, Cat. #C0130, C7657, H3506, and D4513, respectively) at 900 rpm at 37°C for 30 minutes using ThermoMixer® C (Eppendorf). Hearts were subsequently homogenized through a FisherbrandTM Cell Strainers. After dead cell staining with Live/Dead Fixable Aqua Dead Cell Stain Kit (Invitrogen, Cat. #L34957), single cell suspension of heart tissue was incubated with fluorochrome-conjugated antibodies for 20 minutes at room temperature. A total of 6,000 macrophages (CD45.2+Ly6G-CD64+RFP+) or 800 cardiac neutrophils (CD45.2+Ly6G+CD64-RFP+) were sorted in the same setting as blood neutrophils. RNA extraction, RNA library preparations, sequencing reactions, and initial bioinformatics analysis were conducted at Genewiz, LLC. Total RNA was extracted using RNeasy Plus Mini Kit (Qiagen). RNA was quantified using Qubit Fluorometer (Life Technologies) and RNA integrity was checked with TapeStation (Agilent Technologies). SMART-Seq v4 Ultra Low Input Kit for Sequencing was used for full-length cDNA synthesis and amplification (Clontech), and Illumina Nextera XT library was used for sequencing library preparation. Briefly, cDNA was fragmented, and adaptor was added using Transposase, followed by limited-cycle PCR to enrich and add index to the cDNA fragments. The final library was assessed with Agilent TapeStation. The sequencing libraries were multiplexed and clustered on one lane of a flowcell.