Illumina NovaSeq 6000 sequencing; Mapping the developing human immune system across organs - scRNA-seq
Mapping the developing human immune system across organs - scRNA-seq
All human developmental tissue samples used for this study were obtained from the MRC–Wellcome Trust-funded Human Developmental Biology Resource (HDBR; http://www.hdbr.org) with written consent and approval from the Newcastle and North Tyneside NHS Health Authority Joint Ethics Committee (08/H0906/21+5).All tissues were processed into single cell suspensions immediately upon receipt. Tissue was first minced in a tissue culture dish using scalpel. It was then digested with type IV collagenase (final concentration of 1.6 mg/ml; Worthington) in RPMI (Sigma-Aldrich) supplemented with 10% fetal bovine serum (FBS; Gibco), at 37°C for 30 min with intermittent agitation. Digested tissue was then passed through a 100-µm cell strainer and cells were pelleted by centrifugation at 500 g for 5 min at 4°C. Cells were then resuspended in 5 ml red blood cell lysis buffer (eBioscience) and left for 5-10 min at room temperature. It was then topped up with flow buffer (PBS containing 2% (v/v) FBS and 2 mM EDTA) to 45 ml prior to cell counting and antibody staining. Single cell suspensions were generated from 65 samples across yolk sac, liver, bone marrow, spleen, thymus, kidney and skin of 21 donors. The ages of the donors spanned from 4 pcw (post conception weeks) to 17 pcw.Dissociated cells were stained with anti-CD45 antibody and DAPI prior to sorting. Sorting by flow cytometry was performed with BD FACSAria Fusion Flow Cytometer. CD45 positive fraction was sorted from DAPI negative CD45 positive gate and CD45 negative fraction was sorted from DAPI negative CD45 negative gate. CD45 gating was contiguous so that no live cells were lost in sorting. All tissues were processed into single cell suspensions immediately upon receipt. Tissue was first minced in a tissue culture dish using scalpel. It was then digested with type IV collagenase (final concentration of 1.6 mg/ml; Worthington) in RPMI (Sigma-Aldrich) supplemented with 10% fetal bovine serum (FBS; Gibco), at 37°C for 30 min with intermittent agitation. Digested tissue was then passed through a 100 µm cell strainer and cells were pelleted by centrifugation at 500g for 5 min at 4°C. Cells were then resuspended in 5 ml of red blood cell lysis buffer (eBioscience) and left for 5-10 min at room temperature. It was then topped up with flow buffer (PBS containing 2% (v/v) FBS and 2 mM EDTA) to 45 ml prior to cell counting and antibody staining. B cells were gated from single cell suspensions, then DAPI–CD3–CD20+. The top 1% of cells expressing the highest level of CD38 were gated out to exclude possible plasma cell contamination (which should also be CD20 low and therefore not gated in). The rest of the B cells were then sorted into four fractions: CCR10hi, CCR10loCD27+CD43+, CCR10loCD27–CD43+, and CCR10loCD27–CD43–. CD27 and CD43 gatings were chosen based on fluorescence minus one (FMO) controls. The cells were sorted into RPMI supplemented with 10% FBS, penicillin–streptomycin (Gibco) and glutamax (Thermo Fisher Scientific). For scRNA-seq experiments, either Chromium single cell 3' reagent kit or Chromium single cell V(D)J kits from 10x Genomics were used. Unsorted, or DAPI-CD45+ or DAPI-CD45- FACS-isolated cells were loaded onto each channel of the Chromium chip following the manufacturer's instructions before droplet encapsulation on the Chromium controller. Single-cell cDNA synthesis, amplification, gene expression (GEX) and targeted B cell receptor (BCR) and T cell receptor (TCR) libraries were generated. Targeted enrichment for γδTCR was done following the TCR enrichment protocol from 10x with customised primers binding to the constant region of the TRD and TRG genes as described in Mimitou et al. 2019.