title: GSM7795177: Sample_12_Female_Neutrophil_APOE3_4_CN; Homo sapiens; RNA-Seq
library_construction_protocol: Blood was drowned in CPT tubes (BD Bioscience) and centrifuged at 1700g for 30min, to pellet polymorphonuclear leukocytes (neutrophils) below the gel barrier. Following the removal of plasma and peripheral blood mononuclear cells (PBMCs) from the fraction above the gel barrier, a syringe attached to an 18-gauge needle was used to extract the erythrocyte/neutrophil fraction below the gel as previously described. Cells collected in a 50ml tube were washed in PBS and centrifuged at 600g for 5min at 4°C. Cell pellet was resuspended twice in 10ml of ACK (ammonium chloride potassium) to lyse erythrocytes at RT and washed with PBS. Cells were stained with FITC anti-CD66b (1:100, Biolegend, 305103) in PBS supplemented with 2% fetal bovine serum (FBS) for 20min at 4°C. After staining, cells were washed and sorted using BD FACSAriaTM II (BD Bioscience). Smart-Seq2 libraries were prepared by the Broad Technology Labs and sequenced by the Broad Genomics Platform. cDNA libraries were generated from sorted cells using the Smart-seq2 protocol. RNA sequencing was performed using Illumina NextSeq500 using a High Output v2 kit to generate 2× 25bp reads.