Using TRIP for genome-wide position effect analysis in cultured cells

被引：31

作者：

Akhtar, Waseem ^{[1
]}

Pindyurin, Alexey V. ^{[2
,3
]}

de Jong, Johann

Pagie, Ludo ^{[2
]}

ten Hoeve, Jelle ^{[4
]}

Berns, Anton ^{[1
,5
]}

Wessels, Lodewyk F. A. ^{[4
,6
]}

van Steensel, Bas ^{[2
]}

van Lohuizen, Maarten ^{[1
,7
]}

机构：

[1] Netherlands Canc Inst, Div Mol Genet, NL-1066 CX Amsterdam, Netherlands

[2] Netherlands Canc Inst, Div Gene Regulat, NL-1066 CX Amsterdam, Netherlands

[3] Russian Acad Sci, Inst Mol & Cellular Biol, Siberian Branch, Novosibirsk, Russia

[4] Netherlands Canc Inst, Div Mol Carcinogenesis, Amsterdam, Netherlands

[5] Skolkovo Inst Sci & Technol, Skoltech Ctr Stem Cell Res, Skolkovo, Russia

[6] Delft Univ Technol, Delft Bioinformat Lab, Delft, Netherlands

[7] Canc Genom Ctr, Utrecht, Netherlands

来源：

NATURE PROTOCOLS | 2014年 / 9卷 / 06期

基金：

欧洲研究理事会;

关键词：

ENHANCER TRAP; TRANSPOSON INSERTIONS; GENE-EXPRESSION; SLEEPING-BEAUTY; PIGGYBAC; DNA; DOMAINS; HETEROCHROMATIN; MELANOGASTER; POLYMERASE;

D O I：

10.1038/nprot.2014.072

中图分类号：

Q5 [生物化学];

学科分类号：

071010 ; 081704 ;

摘要：

he influence of local chromatin context on gene expression can be explored by integrating a transcription reporter at different locations in the genome as a sensor. Here we provide a detailed protocol for analyzing thousands of reporters integrated in parallel (TRTRIP) at a genome-wide level. TRTRIP is based on tagging each reporter with a unique barcode, which is used for independent reporter expression analysis and integration site mapping. Compared with previous methods for studying position effects, TRTRIP offers a 100-1,000-fold higher throughput in a faster and less-labor-intensive manner. The entire experimental protocol takes similar to 42 d to complete, with high-throughput sequencing and data analysis requiring an additional similar to 11 d. TRTRIP was developed by using transcription reporters in mouse embryonic stem (mESES) cells, but because of its flexibility the method can be used to probe the influence of chromatin context on a variety of molecular processes in any transfectable cell line.

引用

页码：1255 / 1281

页数：27

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