Illumina HiSeq 4000 paired end sequencing; scRNA-seq of human fetal thymus
The tissue sample used for this study was obtained with written informed consent from the participant in accordance with the guidelines in The Declaration of Helsinki 2000. The human fetal tissue was obtained from the MRC/Wellcome Trust-funded Human Developmental Biology Resource (HDBR, http://www.hdbr.org) with appropriate maternal written consent and approval from the Newcastle and North Tyneside NHS Health Authority Joint Ethics Committee (08/H0906/21+5). HDBR is regulated by the UK Human Tissue Authority (HTA; www.hta.gov.uk) and operates in accordance with the relevant HTA Codes of Practice. The tissue was processed immediately after isolation using enzymatic digestion. Tissue was transferred to a sterile 10mm2 tissue culture dish and cut into <1mm3 segments before being transferred to a 50mL conical tube. 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 (FBS; 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. 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 cells (RBC lysis buffer (eBioscience) for 5 minutes at room temperature and washed once with a flow buffer (PBS containing 5%(v/v) FBS and 2mM EDTA) prior to cell counting. scRNA-seq targeting 5,000 cells per sample was performed using the Chromium Controller (10x Genomics). Single-cell cDNA synthesis, amplification, and sequencing libraries were generated using the Single Cell 5' Reagent Kit following the manufacturer's instructions.