2024-12-09
ENA first public
ERC000011
ENA-CHECKLIST
School of Infection & Immunity,  University of Glasgow,  United Kingdom
INSDC center name
public
INSDC status
ERS20305134
SRA accession
E-MTAB-14191_2:Library 2-4
Submitter Id
ArrayExpress
broker name
cultured ST DC and PB T-Cell
cell_type
not collected
collection date
human
common name
Protocols: Autologous memory CD4pos T-cells from Active or Remission RA were FACS-sorted from PBMCs based on their co-expression of CD3, CD4 and CD45RO. We used CD3 activating antibody to mimic TCR engagement. ST-DC DC2 or DC3 subsets and T-cells were sorted into FACS tubes containing complete RPMI1640 medium (10% FCS, penicillin/streptomycin 100U/mL, and 2mM Glutamax). Co-culture was set up when synovial biopsy yielded sufficient numbers of cells at least in one ST-DC subset (minimal 200 cells). Cells were co-cultured at a 1:5 ratio for each DC subset (200-1000 cells) plus memory T-cell (1000-4000 cells) in 200l of X-VIVO 15 Serum-free Cell Medium (BE02-060F, Lonza) supplemented with 2% human serum (H3667, Sigma) or in complete RPMI1640 in a 96-well round-bottom cell-culture plate (3799 – SLS, Corning). After 5 days in culture, the changes in T-cell phenotype were investigated using scRNAseq (BD Rhapsody Immune Response Panel, described in the section below). Cells for sequencing were isolated from culture wells by gentle pipetting prior to downstream preparation. Cells from from each culture condition were labelled with unique sample identifier tags using the BD Hu Single-Cell Sample Multiplexing Kit (633781/BD Bioscience) according to the manufacturer’s protocol:The following sample Tags were used:Sample Tag 1— ATTCAAGGGCAGCCGCGTCACGATTGGATACGACTGTTGGACCGGSample Tag 2—TGGATGGGATAAGTGCGTGATGGACCGAAGGGACCTCGTGGCCGGSample Tag 3—CGGCTCGTGCTGCGTCGTCTCAAGTCCAGAAACTCCGTGTATCCTSample Tag 4—ATTGGGAGGCTTTCGTACCGCTGCCGCCACCAGGTGATACCCGCTSample Tag 5—CTCCCTGGTGTTCAATACCCGATGTGGTGGGCAGAATGTGGCTGGSample Tag 6—TTACCCGCAGGAAGACGTATACCCCTCGTGCCAGGCGACCAATGCSample Tag 7—TGTCTACGTCGGACCGCAAGAAGTGAGTCAGAGGCTGCACGCTGTSample Tag 8—CCCCACCAGGTTGCTTTGTCGGACGAGCCCGCACAGCGCTAGGATSample Tag 9—GTGATCCGCGCAGGCACACATACCGACTCAGATGGGTTGTCCAGGSample Tag 10—GCAGCCGGCGTCGTACGAGGCACAGCGGAGACTAGATGAGGCCCCSample Tag 11—CGCGTCCAATTTCCGAAGCCCCGCCCTAGGAGTTCCCCTGCGTGCSample Tag 12—GCCCATTCATTGCACCCGCCAGTGATCGACCCTAGTGGAGCTAAGCells were then loaded onto the scRNA-seq BD Rhapsody Cartridge using the BD Rhapsody Cartridge Reagent Kit (633731) according to the manufacturer’s protocol. Single-cell cDNA was prepared using the BD Rhapsody cDNA Kit (633773). cDNA preparation was followed by single-cell mRNA and Tag library preparation using BD Rhapsody Targeted mRNA and the Tag Amplification Kit (633774) primers for the BD Rhapsody Immune Response Panel (399 genes: 633750).
description
adult
developmental stage
rheumatoid arthritis
disease
not collected
geographic location (country and/or sea)
not applicable
isolate
Multiplexed as in attached metadata
multiplexing
Homo sapiens
organism
Homo sapiens
organism
blood
organism part
Homo sapiens
scientific_name
Library 2-4
title