title: GSM7321002: AOM/DSS early tumor (w8) blood neutrophils/PMNs 1; Mus musculus; RNA-Seq
library_construction_protocol: For sorting of PMNs or other myeloid cells, we utilized Lyz2EGFP reporter mice with additional staining for Ly6G, Ly6C, CD11b, CD45, and CD64. FACS was performed on a 3-laser sorter FACS Melody and a 6-color laser sorter FACS Aria at the Northwestern Flow Cytometry core. EpCAM+ tumor or epithelial cells as well as Ly6Gneg CD64hi¬¬ macrophages were gated out to avoid RNA contamination from other cell types. CD45+ CD11b+ Ly6Cint Lyz2EGFP Ly6Ghi/int were defined as PMNs/ TANs and sorted directly into RNA Lysis Buffer (RLT plus buffer, Qiagen), supplemented with excess amount of RNaseOUT (10 U/μL, Invitrogen). RNA from PMNs was immediately extracted using the RNeasy Mini Plus Kit (Qiagen) and subjected to RNA integrity (RIN) analysis. For quality control of PMN cell sorting, additional replicates were performed with identical gating strategy and subjected to either more rigorous flow cytometric analyses using additional PMN markers, or using Cytospin and immunofluorescence for imaging. Enrichment of PMN gene sets was performed following RNA sequencing to confirm the transcriptomic identity of the sorted cells as well as validity of downstream bioinformatic analyses The NEBNext Ultra II RNA Library Prep Kit for Illumina (New England Biolabs, Ipswich, MA) was used for library preparation per manufacturer's protocol with the addition of NEBNext rRNA depletion option. Following fragmentation to 200-500 base pairs (bp) by Bioruptor Sonicator (Diagenode), reverse transcription (RT) was performed to convert RNA to cDNA, followed by end-repair, adaptor ligation, and PCR amplification of cDNA libraries per manufacturer's instruction (New England Biolabs, Ipswich, MA). Prior to sequencing, prepared libraries were assessed for fragment sizing on a Bioanalyzer using High Sensitivity DNA chip and re-quantified with Qubit. Library sequencing was performed on an Illumina HiSeq 4000 NGS System (Illumina, San Diego, CA) to generate 50 bp reads with 300 million reads and 30x read depth.