title: GSM3698086: F07: B cell; Homo sapiens; RNA-Seq
library_construction_protocol: Peripheral blood samples were obtained from Type 1 diabetes patient. Cell phenotypes were analyzed using a LSRII multicolor flow cytometer (BD Biosciences). Briefly, single cell suspensions were surface-stained for 20 min on ice with predetermined optimal concentrations of indicated fluorochrome-conjugated antibodies (Key resources Table of the CELL article (Ahmed, et al., 2019)) using established methods (Dai et al., 2015; Martina et al., 2015). Acquired samples (5 x 105 to 1 x 106 live events) were properly compensated using single color stains. Data analysis, gating, and graphical presentation were done using FlowJo software (TreeStar). Doublets were excluded from analysis using FSC-Height versus FSC-Width and SSC-Height versus SSC-Width plots. Multiple specificity controls were used. These included human FcR blocking reagent (Miltenyi Biotec), Fluorescence-Minus One (FMO) for CD5, CD19, TCR, IgD, dump gating, and isotype controls. In addition, when applicable, irrelevant cell types were used as internal biological controls and in the case of in vitro stimulation, we used unstimulated cultures as negative controls. As illustrated in the figure S5A of the CELL article (Ahmed, et al., 2019), the Strategy used to isolate DEs for high-throughput sequencing analysis included the gating of lymphocytes, exclusion of doublets and dead cells followed by using of CD5 and CD19 expression to divide live singlets into Bcon, Tcon cells and DE cells which were identified based on TCR expression among CD5+ CD19+ cells. FACS sorted single cells were processed with the Smart-seq2  protocol (Picelli et al., 2014) with the following modifications. RNA purification was performed prior to reverse  transcription using RNAClean XP beads (Beckman Coulter). cDNA was amplified with 21 PCR cycles  followed by DNA cleanup with AMPure XP beads. Libraries were prepared using the Nextera XT Library Prep  kit (Illumina) using custom barcode adapters.
attributes.tag: GEO Accession
attributes.value: GSM3698086