Illumina HiSeq 4000 paired end sequencing; A cell atlas of human thymic development defines T cell repertoire formation
A cell atlas of human thymic development defines T cell repertoire formation
All tissues were processed immediately after isolation using consistent protocols with variation in enzymatic digestion strength. Tissue was transferred to a sterile 10mm2 tissue culture dish and cut into <1mm3 segments before being transferred to a 50mL conical tube. For mild digestion, tissues were digested with 1.6mg/mL collagenase type IV (Worthington) in RPMI (Sigma-Aldrich) supplemented with 10%(v/v) heat-inactivated fetal bovine serum (Gibco), 100U/mL penicillin (Sigma-Aldrich), 0.1mg/mL streptomycin (Sigma-Aldrich), and 2mM L-Glutamine (Sigma- Aldrich) for 30 minutes at 37°C with intermittent shaking. For stringent digestion, tissue was digested with 0.2 mg/ml Liberase ™ (Roche)/0.125 KU DNase1 (Sigma-Aldrich)/10mM HEPES in RPMI for 30 minutes at 37°C with intermittent shaking. The dissociated cells were separated and remaining undigested tissues were digested again with fresh media. This procedure was repeated until the tissue were completely dissociated. Digested tissue was passed through a 100μm filter, and cells collected by centrifugation (500g for 5 minutes at 4°C). Cells were treated with 1X red blood cell (RBC lysis buffer (eBioscience) for 5 minutes at room temperature and washed once with flow buffer (PBS containing 5%(v/v) FBS and 2mM EDTA) prior to cell counting. Isolated thymus cells were stained with a panel of antibodies prior to sorting based on a CD45 or CD3 expression gate. The anti-human monoclonal antibodies used for flow cytometry based immunophenotyping and FAC sorting are listed in Table S1. An antibody cocktail was freshly prepared by adding 3μL of each antibody in 50μL Brilliant Stain Buffer (BD) per tissue. Cells (≤10x106) were resuspended in 50-100μL flow buffer and an equal volume of antibody mix was added to cells from each tissue. Cells were stained for 30 minutes on ice, washed with flow buffer and resuspended at 10x106 cells/mL. Immediately prior to sorting, DAPI (Sigma-Aldrich) was added to a final concentration of 3μM and cells strained through a 35μm filter. Flow sorting was performed on a BD FACSAria™ Fusion instrument using DIVA V8, and data analysed using FlowJo V10.4.1. Cells were gated to remove dead cells and doublets, and then sorted for 10X or SS2 scRNAseq analysis. For 10X droplet microfluidic analysis, cells were sorted into chilled FACS tubes coated with FBS and prefilled with 500μL sterile PBS. Paediatric samples were sorted into 50% FCS and 50% IMDM medium (supplemented with 1% glu, 1%P/S and 10% FCS). Enrichment of EPCAM positive cells were performed using CD326 (EPCAM) microbeads (Miltenyl Biotec., 130-061-101) according to manufacturer's protocol. CD45 depleted cells were obtained using CD45 microbeads (Miltenyl Biotec., 130-045-801) according to manufacturer's protocol. Cell number and viability were checked after the enrichment to ensure that no significant cell death has been caused by the process. For the droplet-encapsulation scRNA-seq experiments, 8000 live, single, CD45+ or CD45- FACS isolated cells or MACS-enriched cells were loaded on to each of the Chromium Controller (10x Genomics). Single cell cDNA synthesis, amplification and sequencing libraries were generated using the Single Cell 3' and 5' Reagent Kit following the manufacturer's instructions.
Experimental Factor: developmental stage
prenatal
Experimental Factor: age
9
Experimental Factor: phenotype
total cells
Experimental Factor: protocol
gene expression