概要: As organisms age; cells accumulate genetic and epigenetic changes that eventually lead to impaired organ function or catastrophic failure such as cancer. Here we describe a single-cell transcriptome analysis of 2544 human pancreas cells from donors; spanning six decades of life. We find that islet cells from older donors have increased levels of disorder as measured both by noise in the transcriptome and by the number of cells which display inappropriate hormone expression; revealing a transcriptional instability associated with aging. By analyzing the spectrum of somatic mutations in single cells from previously-healthy donors; we find a specific age-dependent mutational signature characterized by C to A and C to G transversions; indicators of oxidative stress; which is absent in single cells from human brain tissue or in a tumor cell line. Cells carrying a high load of such mutations also express higher levels of stress and senescence markers; including FOS; JUN; and the cytoplasmic superoxide dismutase SOD1; markers previously linked to pancreatic diseases with substantial age-dependent risk; such as type 2 diabetes mellitus and adenocarcinoma. Thus; our single-cell approach unveils gene expression changes and somatic mutations acquired in aging human tissue; and identifies molecular pathways induced by these genetic changes that could influence human disease. Also; our results demonstrate the feasibility of using single-cell RNA-seq data from primary cells to derive meaningful insights into the genetic processes that operate on aging human tissue and to determine which molecular mechanisms are coordinated with these processes.
项目整体设计: Examination of single cells from primary human pancreas tissue
Single-cells were collected in lysis buffer in 96-well plates, followed by reverse transcription with template-switch using an LNA-modified template switch oligo to generate cDNA. After 21 cycles of pre-amplification, DNA was purified and analyzed on an automated Fragment Analyzer (Advanced Analytical).
建库方案:
Each cell’s cDNA fragment profile was individually inspected and only wells with successful amplification products (concentration higher than 0.06 ng/ul) and with no detectable RNA degradation were selected for final library preparation. Tagmentation assays and barcoded sequencing libraries were prepared using Nextera XT kit (FC-131-1024; Illumina) according to the manufacturer’s instructions. Barcoded libraries were pooled and subjected to 75 bp paired-end sequencing on the Illumina NextSeq instrument.
测序信息
分子类型:
poly(A)+ RNA
库的片段类型:
PAIRED
库的链类型:
-
测序平台:
ILLUMINA
测序仪型号:
Illumina NextSeq 500
链特异性:
Unspecific
样本
基本信息:
样本描述:
生物条件:
实验变量:
方案:
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质量评估:
数据来源
GEN样本编号
GEN数据集编号
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样本编号
样本名称
生物样本编号
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释放时间
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物种
种族
族裔
年龄
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性别
来源名称
组织
细胞类型
细胞亚型
细胞系
疾病
疾病状态
发育阶段
突变/变异
表型
Condition Detail
生长方案
处理方案
提取方案
建库方案
分子类型
库的片段类型
链特异性
库的链类型
加标（Spike-In）
测序方法
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细胞数
测序片段数
碱基数
平均测序片段长度_1
平均测序片段长度_2
唯一比对率
多重比对率
覆盖度
文章
Single-Cell Analysis of Human Pancreas Reveals Transcriptional Signatures of Aging and Somatic Mutation Patterns.
