title: GSM7818740: Mouse neutrophil subset,OTT BLD,IMM, replicate 3; Mus musculus; ATAC-seq
library_construction_protocol: Tumor: At 6 weeks post injection, tumors were excised from tumor-bearing mice and weighed before mincing into smaller pieces using surgical scissors. Digestion mix (RPMI, 10% FCS, 500U/mL DNase I, 385U/mL collagenase D) was added to the minced tumor and incubated at 37°C for 30 minutes. Digestion enzymes were quenched with FACS buffer, and minced tumors were transferred into the gentleMACS™ C tubes (Miltenyi Biotec) for mechanical homogenization by the Xiril Dispomix using 2 rounds of Program 5 (20 seconds). Homogenized cells were filtered through 70-µm nylon mesh filter and washed with 20mL FACS buffer. Samples were then spun down at 400g for 5 minutes before RBC lysis with 1 X RBC lysis buffer for 3 minutes. Cells were washed filtered through a 70-µm nylon mesh filter. Blood: Murine blood was collected via cardiac puncture or incision in the submandibular region using a 5mm single-use lancet. 200 µL was transferred into an Eppendorf tube containing 20 µL of 0.5M EDTA. Blood samples were then lysed in 1 X red blood cell (RBC) lysis buffer (eBioscience) for 4 min. Samples were quenched with fluorescence-activated cell sorting (FACS) Buffer (1 X PBS, 4mM EDTA, 3% FCS) and spun down at 400g for 5 minutes. RBC lysis was repeated for a second time for another 2 minutes. Spleen: Spleens were excised from mice and single-cell suspensions were obtained by homogenizing spleen samples through a 70-µm nylon mesh filter with FACS buffer. Samples were then spun down at 400g for 5 minutes and lysed with 1 X RBC lysis buffer for 3 minutes. Cells were quenched with FACS buffer and filtered through a 70-µm nylon mesh filter. Bone marrow: A 23-gauge needle through the marrow cavity of the femur and the bone marrow was flushed out with 2 ml of FACS buffer using a 1ml syringe. For sorting purposes that required large cell numbers, both femur and tibia bones were used. Cells were then filtered and washed with FACS buffer, cells were spun down at 400g for 5 minutes. Sorting and hashtag labelling: Total neutrophils were sorted from the bone marrow, spleen, blood, and tumors from pancreatic tumor mice and identified as Lin-CD45+CD115-Ly6ClowSiglec-F-Gr1+CD11b+Ly6G+. Mature neutrophils were further identified as CD101+ while immature neutrophils were CD101-. ATAC-seq was performed based on the Omni-ATAC protocol. 50,000 cells per condition were sorted, washed in FACS buffer, and pelleted before lysis for 3 minutes in Lysis Buffer containing 0.1% v/v NP-40, 0.1% v/v Tween-20 and 0.01% v/v Digitonin. Nuclei were then pelleted and transposed with Tn5 (Illumina) at 37oC, for 30 minutes on a thermomixer at 1000 rpm, before DNA purification, indexing and library preparation was carried out. Samples were checked for nucleosomal patterning via Bioanalyzer before pooling at an equimolar ratio for sequencing on a HiSeqX (Illumina) with paired-end 150 reads.