An optimized approach for multiplexing single-nuclear ATAC-seq using oligonucleotide-conjugated antibodies.
An optimized approach for multiplexing single-nuclear ATAC-seq using oligonucleotide-conjugated antibodies.
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DOI：
10.1186/s13072-023-00486-7
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发表时间：
2023-04-28
影响因子：
3.9
通讯作者：
中科院分区：
文献类型：
--
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单细胞转录和染色质结构分析技术已被广泛应用于许多研究领域，以单细胞分辨率揭示细胞的功能和分子特性。当进行单细胞分析时，样品多路复用技术是有价值的，减少了技术变化并允许成本效益。在许多scRNA-seq研究中已经使用了几种市售方法。另一方面，虽然已经公布了几种方法，但用于转座酶可及染色质（snATAC）-seq测定的单核测定的多重技术仍在开发中。我们开发了一种简单的核哈希方法，使用识别核孔复合物蛋白的寡核苷酸缀合抗体NuHash，通过多重方法进行snATAC-seq库制备。我们使用NuHash对人和小鼠细胞样品的混合物（两个样品，2重，和四个样品，4重）进行多重snATAC-seq分析。对具有至少10，000个读段计数的细胞核的分析显示，NuHash的解复用准确性很高，并且9144个细胞核中只有10个（2重）和12，208个细胞核中只有150个（4重）在NuHash解复用和使用参考基因组比对的区分之间具有不一致的分类。女性和男性样本之间的差异开放染色质区域（OCR）分析显示，男性特异性OCR富集在Y染色体（9个中的4个）。我们还发现5个女性特异性OCR（20个OCR）位于X染色体上。对相同样品的snATAC-seq和深度测序的本体ATAC-seq之间的比较分析揭示，本体ATAC-seq信号强度与snATAC-seq中检测到的细胞簇的数目正相关。此外，当我们基于峰存在的细胞簇的数量对snATAC-seq峰进行分类时，我们观察到组之间不同基因组特征的不同分布。该结果表明，本体ATAC-seq的峰强度可用于鉴定不同类型的功能基因座。我们使用寡聚缀合的抗核孔复合物蛋白质NuHash的多路复用方法允许样品的高精度多路分解。NuHash协议简单明了，适用于冷冻样品，并且不需要修改snATAC-seq文库制备。在线版本包含补充材料，可通过10.1186/s13072-023-00486-7获得。
Single-cell technologies to analyze transcription and chromatin structure have been widely used in many research areas to reveal the functions and molecular properties of cells at single-cell resolution. Sample multiplexing techniques are valuable when performing single-cell analysis, reducing technical variation and permitting cost efficiencies. Several commercially available methods have been used in many scRNA-seq studies. On the other hand, while several methods have been published, multiplexing techniques for single nuclear assay for transposase-accessible chromatin (snATAC)-seq assays remain under development. We developed a simple nucleus hashing method using oligonucleotide-conjugated antibodies recognizing nuclear pore complex proteins, NuHash, to perform snATAC-seq library preparations by multiplexing. We performed multiplexing snATAC-seq analyses on a mixture of human and mouse cell samples (two samples, 2-plex, and four samples, 4-plex) using NuHash. The analyses on nuclei with at least 10,000 read counts showed that the demultiplexing accuracy of NuHash was high, and only ten out of 9144 nuclei (2-plex) and 150 of 12,208 nuclei (4-plex) had discordant classifications between NuHash demultiplexing and discrimination using reference genome alignments. The differential open chromatin region (OCR) analysis between female and male samples revealed that male-specific OCRs were enriched in chromosome Y (four out of nine). We also found that five female-specific OCRs (20 OCRs) were on chromosome X. A comparative analysis between snATAC-seq and deeply sequenced bulk ATAC-seq on the same samples revealed that the bulk ATAC-seq signal intensity was positively correlated with the number of cell clusters detected in snATAC-seq. Moreover, when we categorized snATAC-seq peaks based on the number of cell clusters in which the peak was present, we observed different distributions over different genomic features between the groups. This result suggests that the peak intensities of bulk ATAC-seq can be used to identify different types of functional loci. Our multiplexing method using oligo-conjugated anti-nuclear pore complex proteins, NuHash, permits high-accuracy demultiplexing of samples. The NuHash protocol is straightforward, works on frozen samples, and requires no modifications for snATAC-seq library preparation. The online version contains supplementary material available at 10.1186/s13072-023-00486-7.
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影响因子：
48
作者：
McGinnistm, Christopher S.;Patterson, David M.;Gartner, Zev J.
通讯作者：
Gartner, Zev J.
影响因子：
12.3
作者：
Huang, Yuanhua;McCarthy, Davis J.;Stegle, Oliver
通讯作者：
Stegle, Oliver
影响因子：
16
作者：
Wang K;Xiao Z;Yan Y;Ye R;Hu M;Bai S;Sei E;Qiao Y;Chen H;Lim B;Lin SH;Navin NE
通讯作者：
Navin NE
影响因子：
2.8
作者：
Stirling C;Andrews S;Croft T;Vickers J;Turner P;Robinson A
通讯作者：
Robinson A
影响因子：
3.7
作者：
Carroll TS;Liang Z;Salama R;Stark R;de Santiago I
通讯作者：
de Santiago I