NextSeq 500 paired end sequencing; Single cell RNAseq analysis of anti-Vaccinia long-lived memory B cells (CEL-seq2)
Single cell RNAseq analysis of anti-Vaccinia long-lived memory B cells (CEL-seq2)
All spleen in this study were collected from organ donors who died from stroke or head trauma. None of them had lymphoma or autoimmune disease. Immediately after splenectomy, mononuclear cells were isolated from crushed pieces of splenic tissue by ficoll density-gradient centrifugation and stored in liquid nitrogen. After thawing and subsequent apoptotic cell removal step using the Dead Cell Removal kit (Miltenyi), mononuclear cells were counted and stained with antibodies directed against surface markers. Splenocytes were first enriched for IgG+CD3-CD14-CD16+ cells from which B5R-specific and total IgG+ switched memory B cells (B5R+ or B5R-IgG+CD27+CD20+IgD-) were further single-cell FACS-sorted (AriaIII) into 384-well plates containing 384 primers and Mineral oil (Sigma). Naives B cells were sorted from the IgG-CD3-CD14-CD16+ fraction as CD20+CD38lowCD24intCD27-IgD+. After sorting, plates were snap-frozen on dry ice and stored at -80° C. The “well id”, \"cell type\", “additional cell attribute” and “disease” attributes in plate layouts recapitulate all sorting information for each cell. Index-sorting information and parallel in vitro validation using single culture were additionally use to optimized gating strategies and remove potential sort contaminants from further analysis in the context of IgG+ B5R+ memory B cell sorts (“indexsorting.validation” attributes in plate layouts). This dataset was performed in two distincts batches: batch 1 [Samples: EW01 and EW02] and batch 2 [Samples: CW01 to CW10]. Each sample corresponds to a sorted 384 plate containing cells from one donor from all major populations to be compared. “group”, “individual”, “age” and “sex” attributes in plate layouts recapitulate information for each sorted donor as they appear in the original publication. “sort id” attribute indicate unique sort sessions. All plates from each of the two batches, regardless of the sorting date, were processed at the same time for library preparation and sequencing. For amplification, cells were heat-lysed at 65°C followed by cDNA synthesis using the CEL-Seq2 protocol (T. Hashimshony, N. Senderovich, G. Avital, A. Klochendler, Y. de Leeuw, L. Anavy, D. Gennert, S. Li, K. J. Livak, O. Rozenblatt-Rosen, Y. Dor, A. Regev, I. Yanai, CEL-Seq2: sensitive highly-multiplexed single-cell RNA-Seq, Genome Biol. 17, 77 (2016)) and robotic liquid handling platforms. After second strand cDNA synthesis, the barcoded material was pooled into libraries of 384 cells and amplified using IVT. Following amplification, the rest of the CEL-seq2 protocol (T. Hashimshony, N. Senderovich, G. Avital, A. Klochendler, Y. de Leeuw, L. Anavy, D. Gennert, S. Li, K. J. Livak, O. Rozenblatt-Rosen, Y. Dor, A. Regev, I. Yanai, CEL-Seq2: sensitive highly-multiplexed single-cell RNA-Seq, Genome Biol. 17, 77 (2016)) was followed for preparation of the amplified cDNA library, using TruSeq small RNA primers (Illumina).