2024-12-09
ENA first public
ERC000011
ENA-CHECKLIST
School of Infection & Immunity College of Medical, Veterinary and Life Sciences University of Glasgow
INSDC center name
public
INSDC status
ERS20240729
SRA accession
E-MTAB-14169:BioRRA_Study_SequencingExperiment1
Submitter Id
ArrayExpress
broker name
myeloid cell
cell_type
not collected
collection date
human
common name
Protocols: To study the peripheral blood precursors of synovial tissue dendritic cell clusters we used the BioRRA cohort (Baker et al., 2019); patients fulfilling the American College of Rheumatology revised criteria for RA (Aletaha et al., 2010) and in remission (DAS28-CRP< 2.4 and no Power Doppler signal on a 7-joint ultrasound examination) on cDMARDs (methotrexate, sulfasalazine and/or hydroxychloroquine therapy) stopped all DMARD therapy without tapering. Other medications, including non-steroidal anti-inflammatory drugs were continued if required. Study reviews were scheduled at months 1, 3 and 6, with additional study visits if requested by the patient. During each review, the maintenance of remission or the emergence of disease flare (defined as DAS28-CRP ≥ 2.4) was recorded. PBMCs isolated from anticoagulated peripheral blood from n=12 RA patients in sustained disease remission at baseline (at which treatment was withdrawn without tapering) were isolated by density centrifugation and collected into foetal calf serum (FCS) with 10% DMSO and stored at -150°C. On the day of myeloid cell isolation, PBMCs were carefully defrosted and all PB myeloid cells were FACS-sorted based on their absence of markers of T-, B- and NK-cells. Myeloid cells were suspended in FCS with 10% DMSO and 1% penicillin/streptomycin and stored in liquid nitrogen. On the day of scRNAseq preparation, myeloid cells were carefully thawed and resuspended in FACS tubes containing 1ml complete RPMI 1640 medium. Cells which were live (negative for Fixable Viability Dye eFluor® 780 eBioscience), negative for other lineage markers and expressing high levels of HLA-DR were sorted to FACS tubes in 1ml complete RPMI 1640 medium  Live, HLA-DRhigh cells from each sample 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 (baseline and endpoint flare + remission)
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
BioRRA_Study_SequencingExperiment1
title