Inherited PD-1 deficiency: single-cell RNA sequencing (scRNASeq) of PBMCs

To identify transcriptional modules perturbed by inherited PD-1 deficiency, single-cell RNA sequencing was performed on PBMCs obtained from the ten-year-old PD-1-deficient patient and his healthy six-year-old brother (wild-type for the PDCD1 allele). The preparation was enriched in viable cells by resuspending 1×10^6 cells in 200 μL of 2% FBS and 1 mM calcium chloride in PBS and passing the suspension through a strainer with 40 µm pores, before removing dead cells with the EasySep Dead Cell Removal Kit (StemCell Technologies, Cat: 17899). The initial viability of ~50% was increased to 79% and 75% for the samples of the patient and his brother, respectively. The samples were loaded onto a 10X Genomics Chromium chip for single-cell capture. Libraries were prepared with the Chromium Single Cell 3’ Reagent Kit (v3.1). Library quality was assessed with a Bioanalyzer DNA chip. The libraries were sequenced on one lane (S4 flowcell) of an Illumina NovaSeq 6000 sequencer. Sequences were processed with Cell Ranger 3.1.0. The filtered feature-cell matrix was used for subsequent analyses. Separately, PBMCs from four healthy controls were processed and sequenced as described above (i.e., historical controls), and the data were analyzed simultaneously. Publicly available control PBMC datasets were downloaded from the 10X Genomics website (https://support.10xgenomics.com/single-cell-gene-expression/datasets). The filtered feature-cell matrix was retrieved for the following datasets: frozen PBMCs (Donors A to C) from Zheng et al.64, 33,000 PBMCs from a healthy donor from the Chromium demonstration (v1 Chemistry, processed by Cell Ranger 1.1.0), 8,000 PBMCs from a healthy donor from the Chromium demonstration (v2 Chemistry, processed by Cell Ranger 2.1.0), 10,000 PBMCs from a healthy donor from the Chromium demonstration (v3 Chemistry, processed by Cell Ranger 3.0.0) with or without cell surface protein levels determined by TotalSeq, 5,000 PBMCs from a healthy donor from the Chromium Next GEM demonstration (v3.1 Chemistry, processed by Cell Ranger 3.0.2) with or without cell surface protein levels determined by TotalSeq, and a total of 66,000 PBMCs from an aggregate of eight Chromium connect channels and eight manual channels (v3.1 Chemistry, processed by Cell Ranger 3.1.0). These 10 datasets were used as the baseline for subsequent analyses.

为鉴定受遗传性程序性死亡受体1（PD-1）缺陷扰动的转录模块，研究人员对一名10岁PD-1缺陷患者及其健康的6岁兄弟（PDCD1等位基因野生型）的外周血单个核细胞（Peripheral Blood Mononuclear Cell, PBMC）进行了单细胞RNA测序。
为富集活细胞，将1×10^6个细胞重悬于200μL含2%胎牛血清（Fetal Bovine Serum, FBS）和1mM氯化钙的磷酸盐缓冲液（Phosphate Buffered Saline, PBS）中，通过40μm孔径的滤网过滤细胞悬液，随后使用EasySep死细胞去除试剂盒（EasySep Dead Cell Removal Kit, StemCell Technologies, 货号：17899）去除死细胞。初始活细胞率约为50%，经处理后患者样本与兄弟样本的活细胞率分别提升至79%与75%。
将样本加载至10X Genomics Chromium芯片以进行单细胞捕获，使用Chromium单细胞3’试剂试剂盒（v3.1）构建文库。文库质量通过Bioanalyzer DNA芯片进行评估，随后将文库置于Illumina NovaSeq 6000测序仪的S4流动槽泳道上完成测序。测序数据通过Cell Ranger 3.1.0进行处理，所得过滤后的特征-细胞矩阵用于后续分析。
此外，研究人员按照上述流程处理并测序了4名健康对照者的PBMC，即历史对照，并将其数据与本次实验数据同步分析。同时从10X Genomics官网（https://support.10xgenomics.com/single-cell-gene-expression/datasets）下载公开可用的对照PBMC数据集，获取的过滤后特征-细胞矩阵对应如下数据集：Zheng等64研究中的冷冻PBMC（供者A至C）、Chromium演示实验中来自1名健康供者的33000个PBMC（v1化学试剂，经Cell Ranger 1.1.0处理）、Chromium演示实验中来自1名健康供者的8000个PBMC（v2化学试剂，经Cell Ranger 2.1.0处理）、Chromium演示实验中来自1名健康供者的10000个PBMC（v3化学试剂，经Cell Ranger 3.0.0处理，附带或不附带通过TotalSeq检测的细胞表面蛋白水平）、Chromium Next GEM演示实验中来自1名健康供者的5000个PBMC（v3.1化学试剂，经Cell Ranger 3.0.2处理，附带或不附带通过TotalSeq检测的细胞表面蛋白水平），以及由8个Chromium Connect通道和8个手动通道汇总得到的共66000个PBMC（v3.1化学试剂，经Cell Ranger 3.1.0处理）。上述10个数据集被用作后续分析的基线对照。