title: GSM8703689: sorted cells from 4 patients with MIS-C_5-8_hashtags; Homo sapiens; OTHER
library_construction_protocol: Frozen PBMC from 11 MIS-C patients and from 4 patients who were 6 weeks post-infection with SARS-CoV-2 without developing MIS-C were thawed in RPMI 1640 with 20%v/v FCS. Cells were washed once in PBS containing 1%w/v BSA, 2mM EDTA and 2µg/ml Actinomycin D. Cells were incubated with human FcR blocking reagent and stained for 30 min at 4°C with fluorophore-coupled anti-human antibodies. Simultaneously, cells were incubated with DNA barcoded antibodies for Cellular Indexing of Transcriptomes and Epitopes by Sequencing (CITE-seq). DAPI was added before sorting to allow dead cell exclusion. Activated T cells were identified as DAPI-, CD14-, CD19-, CD3+, CD38+ and HLA-DRhigh, memory B cells were identified as DAPI-, CD14-, CD3-, CD19+, CD27+ (and CD38high for plasmablasts), and monocytes were identified as DAPI-, CD14+. For identifying methylprednisolone effects on activated cells For identifying methylprednisolone effects on activated cells, instead of plasmablasts, lineage- HLA-DRhigh cells were sorted (identified as DAPI-, CD14-, CD19-, CD3-, and HLA-DRhigh). After sorting of the cells using FACS technology, the cells were then applied to the 10X Genomics platform using the Single Cell 5' Library & Gel Bead Kit (v2, dual index) following the manufacturer's instructions. Some PBMC were first stimulated with virus-specific peptide-pools for 16 hours in the presence of blocking antibodies for CD28 and CD40. These cells were sorted for expression of DAPI- and dump- (CD14 and CD19) and CD3+, CD154+ and CD69+. The purified cDNA was amplified and then used for 5' gene expression (GEX), CITE-Seq and TCR library preparation using the Single Cell 5' Library & Gel Bead Kit (v2, 10x Genomics). TCR target enrichment was achieved using the Chromium Single Cell V(D)J Enrichment Kit for Human T cells. Through fragmentation, adapter ligation and index PCR the final GEX and TCR libraries were obtained. The CITE-Seq libraries were obtained only by index PCR. The quality of single cell 5' GEX, CITE-Seq and BCR libraries was assessed by Qubit quantification and Bioanalyzer fragment analysis (HS DNA Kit, Agilent). The sequencing was performed on a NextSeq2000 (or NextSeq500) device (Illumina) using NextSeq 1000/2000 P3 reagent kits (200 Cycles, Illumina) with the recommended sequencing conditions for 5' GEX and Cite-Seq libraries (read1: 26nt, read2: 90nt, index1: 10nt, index2: 10nt) and NextSeq 1000/2000 P3 reagent kits.