GSM9664302: 007_NODscid_me4J_BM_Neu_Rep2; Mus musculus; RNA-Seq
Bone marrow neutrophils were isolated by FACS based on co-expression of CD11b and Ly6G. CD11b+Ly6G+ cells were sorted using the BD FACSAria II at the Jackson Laboratory Flow Cytometry Core Facility (Bar Harbor, ME) and collected in FACS buffer. Cells were homogenized in TRIzol Reagent (Thermo Fisher Scientific) by needle shearing, and total RNA was isolated using the miRNeasy Mini Kit (Qiagen) following the manufacturer's protocol, including the optional on-column DNase digestion step to remove residual genomic DNA. RNA concentration and purity were assessed using a NanoDrop 2000 spectrophotometer (Thermo Scientific), and RNA integrity was evaluated using the Total RNA Pico Bioanalyzer Assay on an Agilent 2100 Bioanalyzer (Agilent Technologies). Stranded mRNA sequencing libraries were constructed using the KAPA mRNA HyperPrep Kit (Roche Sequencing and Life Science) following the manufacturer's protocol. Polyadenylated mRNA was enriched from total RNA using oligo-dT magnetic beads, followed by thermal fragmentation and first- and second-strand cDNA synthesis. Illumina-compatible adapters containing unique dual-index barcode sequences were ligated to each library, and libraries were amplified by PCR. Library size distribution and quality were assessed using the D5000 ScreenTape System on an Agilent TapeStation (Agilent Technologies), and library concentration was quantified using the Qubit dsDNA High Sensitivity (HS) Assay on a Qubit Fluorometer (Thermo Fisher Scientific). Sequencing was performed on an Illumina NextSeq 500 using the High Output Reagent Kit v2.5, generating 75 bp single-end reads.