GSM9664296: Plas_NODscid_WT_BM_Neu_IL1A_IL15_Rep2; Mus musculus; RNA-Seq
Bone marrow neutrophils were isolated by negative selection using the Miltenyi Mouse Neutrophil Isolation Kit. Briefly, femurs were flushed with FACS buffer, filtered through a 70 μm cell strainer, and pelleted by centrifugation. The pellet was resuspended in ACK lysis buffer for 10 minutes at room temperature to lyse red blood cells, then washed three times in PBS. Cells were resuspended in neutrophil isolation buffer (PBS supplemented with 1% BSA and 2 mM EDTA) at 250 μL per 5×10⁷ total cells. Neutrophil Biotin-Antibody Cocktail (Miltenyi Biotec, cat. no. 130-097-658) was added at 50 μL per 5×10⁷ cells and cells were incubated at 4°C for 10 minutes. Following a wash, Anti-Biotin MicroBeads were added at 100 μL per 5×10⁷ cells and incubated at 4°C for 15 minutes. Cells were washed, resuspended in 3 mL neutrophil isolation buffer, and applied to an LS column (Miltenyi Biotec, cat. no. 130-042-401) placed in a MidiMACS Separator (Miltenyi Biotec, cat. no. 130-042-301). The unlabeled flow-through containing enriched neutrophils was collected and the column was washed twice with 3 mL neutrophil isolation buffer, yielding 9 mL of total neutrophil suspension. Total RNA was extracted by Plasmidsaurus via automated bead-based methods. Approximately 100,000 cells per sample were submitted in 60 μL of Zymo DNA/RNA Shield. Library preparation was performed by Plasmidsaurus (plasmidsaurus.com) using a proprietary 3' end counting workflow. Polyadenylated mRNA was captured using oligo-dT primers incorporating sample barcodes and unique molecular identifiers (UMIs), followed by second-strand synthesis and fragmentation with adapter incorporation. Single-end sequencing (~90 bp reads) was performed on the Illumina platform, targeting approximately 10 million deduplicated reads per sample. Reads were quality-filtered with FastP v0.24.0, aligned to the reference genome using STAR v2.7.11, and deduplicated by UMI using UMICollapse v1.1.0. Gene-level counts were generated with featureCounts (subread v2.1.1) using strand-specific counting across exons and 3' UTRs. Differential expression was analyzed with edgeR v4.0.16.