Illumina HiSeq 2000 paired end sequencing; scRNA-seq of human Tfh, Tfr and Treg cells sorted from Tonsil, Iliac Lymph Node and PBMCs
scRNA-seq of human Tfh, Tfr and Treg cells sorted from Tonsil, Iliac Lymph Node and PBMCs
Fresh peripheral blood samples were collected from adult healthy volunteers. Tonsils were collected from healthy children submitted to tonsillectomy due to tonsil hypertrophy. Children with any clinical condition, under any drug treatment or submitted to tonsillectomy due to chronic tonsillitis were excluded. Iliac LNs were collected from kidney transplant recipients, prior to transplantation. Patients with immune-mediated disease or under immune-modulatory treatment were excluded. PBMCs were isolated from blood samples by Ficoll-gradient medium (Histopaque-1077, Sigma-Aldrich) using SepMate tubes (StemCell Technologies). Lymphocytes from tonsils and LNs were also isolated by Ficoll-gradient medium after mechanical disruption. Before cell sorting, samples were enriched for CD4+ T cells. Cells were stained with anti-CD127, anti-CD25, anti-CD4, anti-CD45RA, anti-CD45RO, anti-CXCR5, anti-Foxp3, anti-ICOS, anti-PD-1. To obtain mRNA libraries from single cells, Smart-seq2 protocol was performed with minor modifications. In summary, single cells were sorted into 4 μl of Lysis Buffer (0.2% v/v Triton X-100 (Sigma-Aldrich), dNTPs, and ERCC (1:128M dilution) in 96 well plates, spun down and immediately frozen at −80°C. 50U of SMARTScribe Reverse Transcriptase (Clontech) were used for reverse transcription, and 25 cycles were used for cDNA amplification. Sequencing libraries were performed with Nextera XT DNA Sample Preparation Kit (Illumina) using one-fourth of the volumes indicated by Picelli et al.
Experimental Factor: cell type
regulatory T cell
Experimental Factor: single cell identifier
26932_3#125