SARS-CoV-2 infection-induced human B cells

This study was designed to capture the single cell transcriptome and BCR sequence of B cells that were bait sorted using oligo-tagged viral antigens. Human B cells (viable/CD19+/antigen-PE+ or viable/CD19+/antigen-APC+) were bait sorted after initial magnetic enrichment and used in downstream sequencing. PROTOCOLS: PBMCs were isolated from whole blood (acute samples) or leukoreduction filters (convalescent samples) by use of a Lymphoprep Ficoll gradient and any contaminating red blood cells were lysed by ACK buffer. Cells were then frozen at -80 deg C in fetal bovine serum supplemented with 10% DMSO. On the day of sorting and sequencing cells were thawed and B cell were purified by human pan B cell EasySep enrichment kit from STEMCELL technologies. Cells were partitioned into nanoliter-scale Gel Bead-In-EMulsions (GEMs) to achieve single cell resolution for a maximum of 10,000 individual cells per sample. Utilizing the Chromium Single Cell A Chip Kit(10x Genomics, PN-120236) and Chromium Single Cell 5' Library & Gel Bead Kit (10x Genomics, PN-1000006) , poly-adenylated mRNA from an individual cell was tagged with a unique 16 base pair 10x barcode and 10 base pair Unique Molecular Identifier. Libraries were generated using the Chromium Single Cell 5' Feature Barcode Library Kit (10x genomics, 1000080) and the Chromium Single Cell V(D)J Enrichment Kit Human B Cell (10x Genomics, PN-1000016). The cDNA amplicon size for each library was optimized using enzymatic fragmentation and size selection and the concentration and average fragment size of each library was determined using an Agilent High Sensitivity DNA Kit (Agilent). Specifically, user guide CG000186 Rev D was used for all steps. All libraries were sequenced on an Illuina NextSeq550 or an Illumina NextSeq500 with 26 cycles apportioned for read 1, 8 cycles for the i7 index, and 134 cycles for read 2 using an NextSeq 500/550 High Output Kit v2.5 (150 Cycles). Some samples were sequenced using a NextSeq 550 housed within the Pritzker School of Molecular Engineering's Chicago Immunoengineering Innovation Center (Chicago, IL) and others on a NextSeq500 belonging to the University of Chicago Genomics Facility (Chicago, IL - RRID: SCR_019196) DATA PROCESSING PIPELINE: Demultiplexing the Illumina sequencer's base call files (BCLs) for each flowcell directory into FASTQ files was performed by "cellranger mkfastq" (cellranger version 3.02) Gene counts were calculated using "cellranger count" (cellranger version 3.02) under default setting， BCR sequences were assembled using "cellranger vdj" (cellranger version 3.02) under default setting. GRCh38-1.2.0 for transccriptome, cellranger-vdj-GRCh38-alts-ensembl-2.0.0 for V(D)J本研究旨在获取经寡核苷酸标记病毒抗原诱饵分选的B细胞的单细胞转录组与B细胞受体（B Cell Receptor, BCR）序列。本研究使用的人B细胞（活细胞/CD19+/抗原-PE+ 或 活细胞/CD19+/抗原-APC+）在初始磁富集后经诱饵分选，用于后续测序。 实验方案：从全血（急性期样本）或白细胞滤器（恢复期样本）中分离外周血单个核细胞（PBMCs），采用Lymphoprep Ficoll密度梯度离心法，并用ACK裂解液裂解残留红细胞。随后将细胞置于添加10%二甲基亚砜（DMSO）的胎牛血清中，于-80℃冻存。分选与测序当日，将细胞复苏，使用STEMCELL Technologies公司的人全B细胞EasySep富集试剂盒纯化B细胞。将细胞分配至纳升级油包水凝胶珠微滴体系（Gel Bead-In-EMulsions, GEMs）中，以实现单细胞分辨率，每样本最多处理10000个单个细胞。使用Chromium单细胞A芯片试剂盒（10x Genomics，货号PN-120236）与Chromium单细胞5'文库及凝胶珠试剂盒（10x Genomics，货号PN-1000006），将单个细胞内的聚腺苷酸化mRNA标记有独特的16bp 10x条形码与10bp唯一分子标识符（Unique Molecular Identifier, UMI）。采用Chromium单细胞5'特征条形码文库试剂盒（10x Genomics，货号1000080）与Chromium单细胞V(D)J富集试剂盒（人B细胞专用，10x Genomics，货号PN-1000016）构建文库。通过酶切片段化与尺寸选择优化各文库的cDNA扩增子片段大小，并使用安捷伦高灵敏度DNA试剂盒（Agilent）测定各文库的浓度与平均片段大小。所有步骤均遵循用户指南CG000186 Rev D。所有文库均在Illumina NextSeq550或Illumina NextSeq500测序仪上进行测序，使用NextSeq 500/550高产出试剂盒v2.5（150个循环），其中read 1设为26个循环，i7索引设为8个循环，read 2设为134个循环。部分样本于芝加哥大学普利兹克分子工程学院芝加哥免疫工程创新中心（伊利诺伊州芝加哥市）的NextSeq 550测序仪上完成测序，其余样本则由芝加哥大学基因组学设施（伊利诺伊州芝加哥市，RRID: SCR_019196）的NextSeq500测序仪完成测序。 数据分析流程：使用"cellranger mkfastq"（cellranger 3.02版本）将Illumina测序仪每个流动槽目录的碱基识别文件（BCLs）解复用以生成FASTQ文件。使用"cellranger count"（cellranger 3.02版本，默认参数）计算基因计数；使用"cellranger vdj"（cellranger 3.02版本，默认参数）组装BCR序列。转录组分析参考GRCh38-1.2.0版本，V(D)J分析参考cellranger-vdj-GRCh38-alts-ensembl-2.0.0版本。