title: GSM8686934: Donor1, IL27 treated M2; Homo sapiens; RNA-Seq
library_name: GSM8686934
library_construction_protocol: M2 macrophages (4x10^6 cells) were seeded in 60 mm petri dishes and cultured at 37 °C for more than 16 h in D10 media. The cells were polarized with IL-6, BAY60-6583 or IL-27 for 3 days and then washed with the warmed PBS three times followed by incubation with 2.5 mL of trypsin-EDTA (0.25%) (QBI) at 37 °C for 15 min in a CO2 (5%) incubator. After the incubation, 5 mL of warm D10 was added per dish and the cells were detached by flushing the media. Cells were washed three times with D10 media and cell viability, and cell number were determined using Cellometer Auto 20000 (Nexcellom Bioscience, Lawrence, MA) with the ViaStain AOPI Staining Solution (Nexcellom Bioscience).  The cell viability was 98% ~ 100 %. Each cell was resuspended at 1200 cells/µL in D10 The scRNA sequence libraries were constructed using the Chromium Next GEM Single Cell 5' Reagent Kit V2 (Dual Index) (10x Genomics, Pleasanton, CA) following the Vendor's instruction. Briefly, 16.5 µL of cell suspension were mixed with 16.5 µL of master mix containing reverse transcription (RT) reagent and template switch oligos. The single cells generation and barcoding followed by cDNA synthesis was run on the Chromium Chip K in the 10x Chromium Controller (10x Genomics). The cDNA was amplified by 14 cycles reaction. Based on Agilent 2100 Bioanalyzer (Agilent Biotechnology, Santa Clara, CA) and Qubit 4 analysis (Thermo Fisher). The scRNA sequence libraries contained a single peak of DNA between 300-1000bp, with an average fragment size of 400-500bp.