Illumina HiSeq 2500 paired end sequencing; Single cell RNA sequencing of putative AIRE+ dendritic cells from human paediatric tonsillitis
Single cell RNA sequencing of putative AIRE+ dendritic cells from human paediatric tonsillitis
Tonsils were obtained from pediatric tonsillectomies.  Tissues were disrupted by mechanical means and pressed through a stainless steel mesh to obtain a single-cell suspension. Mononuclear cells were isolated from a Ficoll–Hypaque density gradient.  Tonsil mononuclear cells were depleted of T and B cells by FITC-conjugated anti-CD3, anti-CD19 and anti-FITC micro-beads and passed over an LD magnetic column. Cells were subsequently sorted to purity using a FACSAria and antibodies against lineage (CD3, CD19, CD20, CD94, CD34, CD16,CD14, TCRαβ, TCRγδ), HLA-DR, CD11c, CCR7, CD123 and BDCA2. eTACs were sorted as Lin-CD123-BDCA2-HLADR+CCR7+, cDCs as Lin-CD123-BDCA2-HLADR+CD11c+ and pDCs as Lin-HLADR+CD123+BDCA2+. Cells were incubated with an FcR block prior to antibody staining.  Consent was not given to record age and gender information of donors. Single cell cDNA was generated using the SMART-seq2 protocol. Single cell cDNA was generated using the SMART-seq2 protocol.
Experimental Factor: single cell identifier
eTAC_8_207