Ly6C-low and Ly6C-high neutrophils bulk RNA-seq data
Neutrophils (single cells - Ly6G+ cells) from 4T1 tumor bearing mice were sorted based on Ly6C high or low expression and sequenced. Overall design: Ly6C-high and Ly6C-low neutrophils fom 4T1 tumor-bearing mice were sorted. Then total RNA was extracted using a Qiagen RNeasy Plus kit, according to the manufacturer's instructions. We then measured the concentration of the total RNA using Qubit (Thermo Fisher) and Agilent RNA 6000 Nano kit (Cat. ID: 5067-1511) to analyze its quality. Libraries were made using the Kapa mRNA Hyper prep protocol: the mRNA is captured with oligo-dT beads and then fragmented using heat and magnesium. The first cDNA strand is synthesized using random priming. Then, this step is followed by a combined second strand synthesis and A-tailing, which converts the cDNA:RNA hybrid to double-stranded cDNA (dscDNA), incorporates dUTP into the second cDNA strand, and adds dAMP to the 3' ends of the resulting dscDNA. The next steps are Illumina adaptor ligation and then library amplification using high-fidelity, low-bias PCR. The strand marked with dUTP is not amplified, allowing strand-specific sequencing. As starting material, we used 100 ng of total RNA in 50 µl of DNase-free water. For fragmentation, we incubated the samples 6 minutes at 85°C (fragments of 300–400 bp). For PCR enrichment, we used 13 cycles. Quality control for the final libraries was done using Qubit (DNA HS assay) and Bioanalyzer (DNA HS assay), then we pooled the samples and checked the final library using the Kapa qPCR. Sequencing was done using the Illumina NextSeq 500/550 Mid Output Kit v2.5 (~24M single-end 75-cycles reads for each sample).