A practical solution for preserving single cells for RNA sequencing.
A practical solution for preserving single cells for RNA sequencing.
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DOI：
10.1038/s41598-018-20372-7
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发表时间：
2018-02-01
影响因子：
4.6
通讯作者：
Bowden R
中科院分区：
文献类型：
--
作者：
Attar M;Sharma E;Li S;Bryer C;Cubitt L;Broxholme J;Lockstone H;Kinchen J;Simmons A;Piazza P;Buck D;Livak KJ;Bowden R
单细胞实验的设计和实施往往受到其对新鲜起始材料需求的限制。我们采用了一种使用二硫代双（丙酸琥珀酰亚胺酯）（DSP），即洛曼试剂进行组织学组织固定的方法，以稳定细胞样本用于单细胞转录组应用。DSP是一种游离胺基的可逆交联剂，先前已被证明在保持组织学的组织完整性的同时，能在大量RNA提取中维持RNA的完整性和产量。尽管来自DSP固定的单细胞的RNA - seq数据似乎容易出现特征性的人为假象，例如与新鲜细胞相比，cDNA产量略有降低以及可检测到的3′端偏差，但使用DSP进行细胞保存似乎不会在基因水平上大幅降低RNA的复杂性。此外，有证据表明细胞的即时固定可以减少细胞间的技术变异性。DSP固定的细胞能够保留常用的染料，如碘化丙啶，这使得能够追踪实验亚群，并记录固定时的细胞活力。使用DSP保存细胞将消除单细胞实验阶段的几个障碍，包括样本运输以及下游单细胞分离和处理所需共享设备的时间安排。
The design and implementation of single-cell experiments is often limited by their requirement for fresh starting material. We have adapted a method for histological tissue fixation using dithio-bis(succinimidyl propionate) (DSP), or Lomant’s Reagent, to stabilise cell samples for single-cell transcriptomic applications. DSP is a reversible cross-linker of free amine groups that has previously been shown to preserve tissue integrity for histology while maintaining RNA integrity and yield in bulk RNA extractions. Although RNA-seq data from DSP-fixed single cells appears to be prone to characteristic artefacts, such as slightly reduced yield of cDNA and a detectable 3′ bias in comparison with fresh cells, cell preservation using DSP does not appear to substantially reduce RNA complexity at the gene level. In addition, there is evidence that instantaneous fixation of cells can reduce inter-cell technical variability. The ability of DSP-fixed cells to retain commonly used dyes, such as propidium iodide, enables the tracking of experimental sub-populations and the recording of cell viability at the point of fixation. Preserving cells using DSP will remove several barriers in the staging of single-cell experiments, including the transport of samples and the scheduling of shared equipment for downstream single-cell isolation and processing.
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影响因子：
5.4
作者：
Alles J;Karaiskos N;Praktiknjo SD;Grosswendt S;Wahle P;Ruffault PL;Ayoub S;Schreyer L;Boltengagen A;Birchmeier C;Zinzen R;Kocks C;Rajewsky N
通讯作者：
Rajewsky N
影响因子：
14.9
作者：
Flicek P;Amode MR;Barrell D;Beal K;Billis K;Brent S;Carvalho-Silva D;Clapham P;Coates G;Fitzgerald S;Gil L;Girón CG;Gordon L;Hourlier T;Hunt S;Johnson N;Juettemann T;Kähäri AK;Keenan S;Kulesha E;Martin FJ;Maurel T;McLaren WM;Murphy DN;Nag R;Overduin B;Pignatelli M;Pritchard B;Pritchard E;Riat HS;Ruffier M;Sheppard D;Taylor K;Thormann A;Trevanion SJ;Vullo A;Wilder SP;Wilson M;Zadissa A;Aken BL;Birney E;Cunningham F;Harrow J;Herrero J;Hubbard TJ;Kinsella R;Muffato M;Parker A;Spudich G;Yates A;Zerbino DR;Searle SM
通讯作者：
Searle SM
影响因子：
48
作者：
Wu, Angela R.;Neff, Norma F.;Kalisky, Tomer;Dalerba, Piero;Treutlein, Barbara;Rothenberg, Michael E.;Mburu, Francis M.;Mantalas, Gary L.;Sim, Sopheak;Clarke, Michael F.;Quake, Stephen R.
通讯作者：
Quake, Stephen R.
影响因子：
12.3
作者：
Guillaumet-Adkins A;Rodríguez-Esteban G;Mereu E;Mendez-Lago M;Jaitin DA;Villanueva A;Vidal A;Martinez-Marti A;Felip E;Vivancos A;Keren-Shaul H;Heath S;Gut M;Amit I;Gut I;Heyn H
通讯作者：
Heyn H
影响因子：
48
作者：
Kiselev, Vladimir Yu;Kirschner, Kristina;Hemberg, Martin
通讯作者：
Hemberg, Martin