Single-Cell RNA Sequencing Protocol
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Corresponding Organization : United States Public Health Service Commissioned Officers Association
Other organizations : National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institute of General Medical Sciences, Ponce Health Sciences University, National Institute of Allergy and Infectious Diseases, National Institutes of Health, National Heart Lung and Blood Institute, Inova Fairfax Hospital, Adventist HealthCare Shady Grove Medical Center
Variable analysis
- Erythroclear Red Blood Cell Depletion Reagent Kit (STEMCELL Technologies, catalog no. 01738) used for isolating white blood cells
- Cell concentration (1000 cells/μl) used for single-cell gel beads in emulsion (GEMs) preparation
- Chromium Single-Cell 3′ Library and Gel Bead Kit v3.1 (10x Genomics, catalog no. 1000121) used for single-cell cDNAs and libraries preparation
- Single-cell cDNAs and libraries obtained
- PBS with 0.02% bovine serum albumin used for cell washing
- GEM-RT incubation performed in a C1000 Touch thermal cycler with 96-deep well reaction module (Bio-Rad, catalog no. 1851197)
- Single-strand cDNAs purified with DynaBeads MyOne Silane Beads (Thermo Fisher Scientific, catalog no. 37002D)
- Amplified cDNA products cleaned with 0.6X DynaBeads MyOne Silane Beads (Thermo Fisher Scientific, catalog no. 37002D)
- Quality and quantity of cDNAs assessed on 4200 TapeStation (Agilent Technologies) with High Sensitivity D5000 DNA ScreenTape (Agilent, catalog no. 5067-5592)
- Libraries diluted to the same molarity and pooled for sequencing on NovaSeq6000 (Illumina) sequencers
- No positive or negative controls were explicitly mentioned in the provided information.
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