Methods and compositions for cDNA synthesis and single-cell transcriptome profiling using template switching reaction

一种模板转换、DNA链的技术，应用在用于使用模板转换反应进行cDNA合成和单细胞转录组概况分析和组合物领域，能够解决尚未报道cDNA文库收率和平均长度的系统性努力等问题

Inactive Publication Date: 2016-05-11
LUDWIG INST FOR CANCER RES LTD
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  • Abstract
  • Description
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Problems solved by technology

Despite the widespread use of these reactions, systematic efforts to improve cDNA library yield and average length from single-cell quantities have not been reported

Method used

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  • Methods and compositions for cDNA synthesis and single-cell transcriptome profiling using template switching reaction
  • Methods and compositions for cDNA synthesis and single-cell transcriptome profiling using template switching reaction
  • Methods and compositions for cDNA synthesis and single-cell transcriptome profiling using template switching reaction

Examples

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example 1

[0077] method

[0078] Experiments using total RNA

[0079] Using DNA extracted from mouse brain and used for Illumina sequencing The control total RNA provided with the UltraLowRNA kit (Clontech Company) was used for RNA experiments. One microliter of 1 ng / μl solution was used in each experiment and combined with 1 μl of anchored oligo-dT primer (10 mM, 5′-AAGCAGTGGTATCAACGCAGAGTACT 30 VN-3', wherein "N" is any base and "V" is "A", "C", or "G") is mixed with 1 μl of dNTP mixture (10 mM, Fermentas), Denature at 72°C for 3 minutes and place on ice immediately thereafter. Add 7 μl of the first-strand reaction mixture to each sample, the first-strand reaction mixture contains 0.50 μl SuperScriptIIRT (200Uml-1, Invitrogen), 0.25 μl RNase inhibitor (20Uml-1, Clontech Company), 2 μl SuperscriptII first-strand buffer solution (5x, Invitrogen), 0.25 μl DTT (100 mM, Invitrogen), 2 μl betaine (5M, Sigma (Sigma)), 0.9 μl MgCl 2 (100 mM, Sigma), 1 μl TSO (10 μM, a full list of oligo...

example 2

[0103] To improve the profiling of full-length transcriptomes obtained from single cells, a large number of variants (457 experiments in total) of reverse transcription, template-switching oligonucleotides (TSOs), and PCR preamplification were performed in terms of cDNA library yield and length evaluation, and compared these results with commercial Smart-Seq (hereinafter referred to as ) compared with (Table 1). Importantly, improvements were identified that significantly increased cDNA yield and length obtained from 1 ng of total promoter RNA (Table 1).

[0104] Specifically, compared to the commercial Smart-Seq used IIAoligo, only a single guanylate at the 3′ end (rGrG+G) of TSO was exchanged for a locked nucleic acid (LNA) guanylate resulting in a 2-fold increase in cDNA yield (p=0.003, Student's t-test; figure 1 a. Table 2 and image 3 ).

[0105] Additionally, the methyl group donor betaine was found to be associated with higher MgCl 2 Concentration binding had a s...

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Abstract

This application discloses methods for cDN'A synthesis with improved reverse transcription, template switching and preamplification to increase both yield and average length of cDNA libraries generated from individual cells. The new methods include exchanging a single nucleoside residue for a locked nucleic acid (INA) at the TSO 3' end, using a methyl group donor, and / or a MgCb concentration higher than conventionally used. Single-cell transcriptome analyses incorporating these differences have full-length coverage, improved sensitivity and accuracy, have less bias and are more amendable to cost-effective automation. The invention also provides cDNA molecules comprising a locked nucleic acid at the 3'-end, compositions and cDNA libraries comprising these cDNA molecules, and methods for single-cell transcriptome profiling.

Description

[0001] Cross References to Related Applications [0002] This application claims priority to US Provisional Patent Application Serial No. 61 / 869,220, filed August 23, 2014, the contents of which are incorporated herein by reference. technical field [0003] The present invention relates to methods for synthesizing double-stranded cDNA with improved yield and average length, as well as synthetic cDNA molecules and cDNA libraries generated from individual cells. Background technique [0004] Single-cell gene expression analysis holds promise for characterizing cellular heterogeneity, but current methods sacrifice coverage, sensitivity, or throughput. Several methods exist for constructing full-length cDNA from large amounts of RNA, including the capen richment procedure (Maruyama, K. and Sugano, S., Gene 138 , 171–174 (1994); Carninci, P. (Carninci, P.) and Lin Qi, Y. (Hayashizaki, Y.), Enzymological Methods (Meth.Enzymol.) 303, 19–44 (1999); Das, M. et al., Physiol. Genomic...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P19/34C07H21/04
CPCC12Q1/6809C12Q1/6853C12N15/09C12N15/1096C40B40/08C40B50/06C12Q2521/107C12Q2525/117C12Q2537/163C12N15/00C12P19/34C12Q1/6883C12Q2600/158
Inventor 里卡德·桑德伯格西莫内·佩斯里欧麦德·R·法瑞达尼
Owner LUDWIG INST FOR CANCER RES LTD
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