title: GSM5471629: SP WT Ly6G high rep 2; Mus musculus; RNA-Seq
library_construction_protocol: All mice used for RNA isolation were sacrificed within the same period in the morning (between 07.00-08.30) to minimise circadian-induced gene expression changes between samples. RNA was isolated from Ly6GInt, Ly6GHi and Ly6G+ neutrophil populations that had been FACS sorted from the BM, spleen, liver and peripheral blood. Biological replicates for PBS and LPS bulk Ly6G+ neutrophil RNA-Seq are as follows: PBS: peripheral blood Ly6GHi n=4 mice; BM Ly6GInt n=4 mice; BM Ly6GHi n=4 mice; spleen Ly6GInt n=6 mice; spleen Ly6GHi n=7 mice; liver Ly6G+ n=7 mice. LPS: peripheral blood Ly6GInt n=4 mice; peripheral blood Ly6GHi n=4 mice; BM Ly6GInt n=3 mice; BM Ly6GHi n=3 mice; spleen Ly6GInt n=4 mice; spleen Ly6GHi n=4 mice; liver Ly6G+ n=4 mice. Biological replicates for PDAC model bulk Ly6G+ neutrophil RNA-Seq are as follows: BM: WT Ly6GInt n=10 mice; KPflC Ly6GInt n=5 mice; KPC Ly6GInt n=5 mice; KPC Ly6GHi n=5 mice. Spleen: WT Ly6GInt n=8 mice; KPflC Ly6GInt n=4 mice; KPC Ly6GInt n=5 mice. RNA was extracted from sorted populations using an RNeasy Micro Kit (Qiagen). RNA quality was evaluated using an Agilent Bioanalyser using RNA Pico chips. Libraries for cluster generation and DNA sequencing were prepared using a TaKaRa SMARTer Stranded Total RNA-Seq – Pico Input Mammalian kit v2.0 as per the manufacturer's instructions. Quality and quantity of the DNA libraries was assessed on an Agilent 2200 Tapestation (D1000 screentape) and Qubit (Thermo Fisher Scientific) respectively. The libraries were run on the Illumina Next Seq 500 using the High Output 75 cycles kit (2x36 cycles, paired end reads, dual index).