title: NextSeq 500 paired end sequencing; single cell RNAseq of memory B cells in the context of rituximab-treated ITP patients
design: single cell RNAseq of memory B cells in the context of rituximab-treated ITP patients
library_construction_protocol: All ITP patients included in this study were adults, and had ITP diagnosis according to the international guidelines definitions. Patients with underlying immunodeficiency, hepatitis C virus infection, lymphoproliferative disorders, thyroid or liver disease, and defined systemic lupus erythematosus (≥ 4 American Rheumatism Association criteria) were excluded. All patients had persistent (≥ 3 months) or chronic (≥ 12 months) ITP. Complete response was defined as platelet count >100×109/L and no bleeding, and failure as platelet count <30×109/L (61). All relapse cases described in this study correspond to patients that first showed a complete response to RTX treatment. Cases of partial restoration of platelet counts (<100×109/L) were excluded as this is likely linked to the persistence of low levels of autoantibody secretion. All healthy controls (HD) were organ donors who died from stroke or head trauma. None of them had lymphoma or autoimmune disease. All young donors included in the scRNA-seq analysis were sickle cell disease patients who were splenectomized between the age of 4 and 5 years.  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, mononuclear cells were counted and stained with antibodies directed against surface markers. Memory B cells (CD19+CD38-CD24+CD27+IgD-) from HD, RTX failure, RTX relapse and young donors were single-cell FACS-sorted (AriaIII) into 384-well plates containing 384 primers and Mineral oil (Sigma). After sorting, plates were snap-frozen on dry ice and stored at -80° C.  Naïves B cells were sorted as CD19+CD38lowCD24intCD27-IgD+, GC B cells as CD19+CD38intCD24- and PC as CD38hi CD27hi. “well id”, \"cell type\", “additional cell attribute” and “disease” attributes in plate layouts recapitulate all sorting information for each cell.  This dataset was performed in two distincts batches. In a pilot batch [Samples: CW11 to CW14 and MW01/MW02], various B cell populations (Naives B cells, Memory B cells, GC B cells and plasmocytes) were sorted from a limited set of donors. The secondary batch [MW03 to MW17] is focused solely on memory B cells from HD and ITP patients. 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).