GSM9248304: MYDGF KO iNeutrophil; Homo sapiens; RNA-Seq
2e5 iNeutrophils were collected at day 7 following transfer into Media C, spun down, and resuspended in 200 µL of PBS + 0.04% Bovine Serum Albumin (BSA; Sigma-Aldrich). All cells were directly used for library construction in a Chromium controller at the Gene Expression Center of the University of Wisconsin–Madison Biotechnology Center (RRID: SCR_017757). Libraries were constructed according to the Chromium Single Cell 3' Reagent Kit v3.1 user guide (10x Genomics, Pleasanton, CA). Cell concentration and viability were quantified on the Luna-FX7 (Logos Biosystems, Anyang, South Korea) using acridine orange/propidium iodide stain. Cells were loaded onto a Chromium Single Cell Chip G chip for the single cell partitioning GEM reaction on the Chromium iX platform. Next, GEMs were transferred to emulsion safe strip tubes for reverse transcription and cell/transcript barcoding using an Eppendorf MasterCycler Pro thermocycler (Eppendorf, Hamburg, Germany). Following reverse transcription, GEMs were broken, and pooled single cell cDNA was amplified for 13 cycles, purified using SPRIselect beads (Beckman Coulter, Brea, CA), and quantified on a Bioanalyzer 2100 (Agilent, Santa Clara, CA) using the High Sensitivity DNA kit. A portion of the full-length cDNA was fragmented and used to generate cDNA libraries according to standard 10x Genomics workflow.