title: GSM8771155: B-cells GEX donor H1; Homo sapiens; OTHER
library_construction_protocol: The cryopreserved cells were thawed into warm RPMI (Sigma-Aldrich) added with 10% FBS and counted for flow cytometry analysis. At least 500,000 cells viable cells were used per panel and samples with <75% viability were discarded. Cells were stained with Zombie Aqua fixable viability kit on ice for 20 min (Biolegend) to discriminate live and dead cells. After washing, cells were stained with surface markers on ice for 30 min. After washing, cells were fixed and permeabilized with True-Nuclear Transcription Factor Buffer set (Biolegend) per manufacturer's instructions, followed by intracellular staining with anti-Ki-67, Granzyme B and Granzyme K antibodies. Cells were resuspended in FACS buffer and analyzed on a BD FACSAria Fusion (BD Biosciences). Data were analyzed with FlowJo software v.10.10.0. Cells were counted and assessed for viability. From the PBMC, we performed a positive selection of B cells with magnetic beads (human CD19 MicroBeads, Miltenyi Biotec). The flow-through was used for the subsequent negative selection of T cells (Human Pan T Cell Isolation Kit, Miltenyi Biotec). Chromium Next GEM Single Cell V(D)J Reagent Kit, applying the protocol 10x CG000207RevE Lib VDJ Tet B+ LibGEX was used to integrate transcriptional profile with the immune repertoire of individual cells, full-length paired T-cell receptor (TCR) and B-cell immunoglobulin (Ig) transcripts. Samples were thawed following the 10x Genomics demonstrated protocol (Dropwise Media Addition & Sequential Dilution, document CG000447). 5000-20000 cells (depending on donor) were loaded on the 10X Chromium Controller to target capturing half of the loaded cells. The barcoded cDNA was processed to prepare cDNA library and VDJ library. Quality control quantification of cDNA and libraries were performed on the 2100 Bioanalyzer with the High Sensitivity DNA Kit (Agilent) and the Qubit with dsDNA HS Assay Kit (ThermoFisher). Libraries were sequenced on Illumina NovaSeq, at a sequencing depth of 20.000 read pairs per cell for gene expression libraries and 5.000 read pairs per cell for V(D)J enriched libraries.
identifiers.namespace: GEO
identifiers.id: GSM8771155_r1