scRNA-seq of young and aged lungs following influenza A virus infection. scRNA-seq of young and aged lungs following influenza A virus infection

Aging is known to alter the host repsonse to influenza infection. Here, we use single-cell RNA sequencing (scRNA-seq) to identify cellular changes in the lungs of young (16-week-old) and aged (80-week-old) mice following influenza infection. Overall design: Mice were infected with influenza A/PR8/34 (~50 PFU/mouse) to establish acute infection. Infections were performed by intranasal (i.n.) administration under anesthesia as described before (Sun et al., 2009). Mice were then euthanized and the right ventricle was perfused with 10 mL cold DPBS (Corning). The lung lobes were cut into fine pieces and digested at 37 °C with collagenase IV (1 mg/mL; Worthington) for 40 minutes. The tissue pieces were further ground and homogenized in a 70 μm cell strainer. After spinning down, red blood cells were removed by addition of ACK lysis buffer (Lonza) for 10 minutes on ice. Cells were kept in 10% RPMI (Lonza) at all times. Only day 9 post-infection samples were sorted for scRNA-seq. Single-cell suspensions were incubated with antibodies against surface markers for 30 minutes at 4 °C in 10% RPMI, followed by 2 washes in 10% RPMI. Samples were then run on an Aria IIIu cell sorter (BD Biosciences). For day 9 post-infection samples, single-cell suspensions of lung tissue were FACS sorted (see above), and Lin+ cells and Lin– cells from each sample were individually pooled at a 1:1 ratio. For day 3 post-infection samples, cells were not sorted as the ratio of Lin+:Lin– cells was approximately 1:1. Cells were loaded onto the Chromium Controller (10x Genomics), then the 10x Genomics 3’ v2 Reagent Kit (day 9 post-infection samples) or the 10x Genomics 3’ v3.1 Reagent Kit (day 3 post-infection samples) was used to generate cDNA libraries. Barcoded libraries were sequenced on an Illumina NextSeq 500 instrument using a NextSeq 500/550 High Output Kit v2 (150 cycles) (20024907, Illumina) with the following cycle counts: 28 (read 1), 8 (index), and 91 (read 2) (day 9 post-infection samples) or 28 (read 1), 10 (index 1), 10 (index 2), 90 (read 2) (day 3 post-infection samples). Data were demultiplexed and aligned to the mm10 2020-A reference transcriptome (10x Genomics) using Cell Ranger (v6.0, 10x Genomics). Analysis was performed in R using Seurat (v4.0) (Butler et al., 2018), with tidyverse (v1.3.1) used for data organization (Wickham et al., 2019). Receptor-ligand analysis was performed using CellChat (v1.1.3) (Jin et al., 2021) with default settings.

已知衰老会改变宿主对流感感染的应答反应。本研究采用单细胞RNA测序（single-cell RNA sequencing, scRNA-seq）技术，旨在鉴定流感感染后年轻（16周龄）与老年（80周龄）小鼠肺部的细胞变化。
实验整体设计如下：小鼠经甲型流感病毒A/PR8/34株（约50 PFU/只）感染以构建急性感染模型，感染操作参照既往研究（Sun等，2009），采用麻醉下鼻内（intranasal, i.n.）滴注方式。随后对小鼠实施安乐死，经右心室灌注10 mL预冷的DPBS（Corning公司）。将肺叶剪碎后，于37 ℃下用胶原酶IV（1 mg/mL，Worthington公司）消化40分钟。将组织碎块进一步研磨，并通过70 μm细胞筛进行匀浆处理。离心沉淀细胞后，采用ACK裂解液（Lonza公司）于冰上孵育10分钟以去除红细胞。实验全程均将细胞保存于10% RPMI培养基（Lonza公司）中。仅针对感染后第9天的样本进行分选以用于scRNA-seq。将单细胞悬液与表面标志物抗体于4 ℃下在10% RPMI培养基中孵育30分钟，随后用10% RPMI培养基洗涤2次。随后将样本上样至Aria IIIu细胞分选仪（BD Biosciences公司）进行分选。针对感染后第9天的样本，先通过流式细胞术（FACS）分选肺组织单细胞悬液（详见上文），再将每份样本中的Lin+细胞与Lin–细胞以1:1的比例分别混合。而感染后第3天的样本无需分选，因其Lin+细胞与Lin–细胞的比例约为1:1。将细胞上样至Chromium Controller系统（10x Genomics公司），随后分别使用10x Genomics 3' v2试剂试剂盒（感染后第9天样本）或10x Genomics 3' v3.1试剂试剂盒（感染后第3天样本）构建cDNA文库。带条形码的文库在Illumina NextSeq 500测序仪上进行测序，采用NextSeq 500/550 High Output Kit v2（150个循环，货号20024907，Illumina公司），测序循环参数如下：感染后第9天样本为读段1（read 1）28个循环、索引（index）8个循环、读段2（read 2）91个循环；感染后第3天样本为读段1 28个循环、索引1（index 1）10个循环、索引2（index 2）10个循环、读段2 90个循环。使用Cell Ranger软件（v6.0，10x Genomics公司）对数据进行解多路复用，并比对至mm10 2020-A参考转录组（10x Genomics公司）。数据分析在R环境中完成，采用Seurat软件包（v4.0，Butler等，2018），并使用tidyverse工具包（v1.3.1，Wickham等，2019）进行数据整理。受体-配体分析采用CellChat软件包（v1.1.3，Jin等，2021），并使用默认参数设置。