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Home > ÀüÁ¦Ç°º¸±â > Epigenetics > ChIP-seq, Cut&Run-seq (NGS) > [Àû¿ë] ¼Ò·®¼¼Æ÷·ÎºÎÅÍ ChIP-seq ºÐ¼®¹ý

[Àû¿ë] ¼Ò·®¼¼Æ÷·ÎºÎÅÍ ChIP-seq ºÐ¼®¹ý

-

¼Ò·®ÀÇ ¼¼Æ÷ (10,000°³ÀÇ ¼¼Æ÷)·ÎºÎÅÍ ChIP-seq ºÐ¼® ±â¹ý È®¸³

- µ¥ÀÌÅÍ Á¦°ø: Prof. Shirahige Katsuhiko
(The Institute for Quantitative Biosciences (IQB), The University of Tokyo)

¡ß Introduction
Å©·Î¸¶Æ¾¸é¿ªÄ§°­¹ý (ChIP; chromatin immuno precipitation)Àº ¸ñÀû ´Ü¹éÁúÀÌ °áÇյǾî ÀÖ´Â ¿°»öü ¿µ¿ªÀ» ¸é¿ªÄ§°­¹ýÀ¸·Î ³óÃà (enrichment)ÇÏ´Â ±â¼úÀÌ´Ù. Chromatin Immunoprecipitation Sequencing (ChIP-seq)Àº ³óÃàÇÑ ¿µ¿ªÀ» Next Generation Sequencing (NGS)À¸·Î ºÐ¼®ÇÏ´Â ¹æ¹ýÀ¸·Î, ¿¡ÇÇÁ¦³×ƽ½º ¿¬±¸¸¦ À§ÇÑ ÇʼöÀûÀÎ ±â¼úÀÌ´Ù. ChIP-seqÀº »ç¿ëÇÏ´Â ¼¼Æ÷ÀÇ ¼ö°¡ Àû°í ChIPÀ¸·ÎºÎÅÍ È¸¼öµÇ´Â DNAÀÇ ¾çÀÌ ¸Å¿ì Àû¾î, ¹Ì·®ÀÇ ChIP DNA ºÐ¼®¹ýÀ» È®¸³ÇÏ´Â °ÍÀº ¸Å¿ì Áß¿äÇÑ °úÁ¦ÀÌ´Ù. ÀÌ¿¡ µû¶ó, º» ¿¬±¸ÀÚ´Â Å©·Î¸¶Æ¾ ´ÜÆíÈ­ (Chromatin fragmentation)ÀÇ ÃÖÀûÈ­ ¹× ´ÙÄ«¶ó¹ÙÀÌ¿ÀÀÇ SMARTer¢ç ThruPLEX¢ç DNA-seq KitÀ» ÀÌ¿ëÇÏ¿©, ¹Ì·®ÀÇ ChIP DNA-seq ºÐ¼® ±â¹ýÀ» È®¸³ÇÏ¿´´Ù.

¡ß »ç¿ëÁ¦Ç°
SMARTer¢ç ThruPLEX¢ç DNA-seq Kit (Takara)

¡ß Workflow


¡ß NGS Mapping °á°ú
1x104°³ ¶Ç´Â 2.5¡¿105°³ÀÇ ¸Á¸·»ö¼Ò»óÇǼ¼Æ÷ (RPE)¿¡¼­ H3K4me3 Ç×ü¸¦ ÀÌ¿ëÇØ ChIP DNA »ùÇÃÀ» ¾ò¾ú´Ù. ¸é¿ª ħ°­ÇÏÁö ¾ÊÀº ¼¼Æ÷¸¦ ¡°Input¡± »ùÇ÷Πǥ±âÇÏ¿´´Ù. ChIP DNA »ùÇÃÀÇ Àü·®À» ÀÌ¿ëÇØ SMARTer¢ç ThruPLEX¢ç DNA-seq Kit·Î NGS Library¸¦ Á¦ÀÛÇÏ¿© ¿°±â¼­¿­À» ºÐ¼®ÇÏ¿´´Ù. 1x104°³ÀÇ ÀûÀº ¼¼Æ÷ »ùÇÿ¡¼­ duplicate rate°¡ 9.1%·Î È®ÀεǾú°í 2.5¡¿105 °³ ¼¼Æ÷ »ùÇÃÀÇ ½ÇÇè °á°ú¿Í À¯»çÇÏ¿´´Ù.
»ùÇÃ

Total reads

Mapped reads

Unmapped reads

PCR Bias
(duplicate)

Input

31,400,938

26,696,740 (78.7%)

3,087,220 (9.8%)

3,616,978 (11.5%)

1x104 ¼¼Æ÷
(H3K4me3)

30,823,404

21,336,878 (69.2%)

6,697,451 (21.7%)

2,789,075 (9.1%)

2.5x105 ¼¼Æ÷
(H3K4me3)

45,738,702

37,730,704 (82.5%)

4,018,338 (8.8%)

3,989,660 (8.7%)


¡ß Peak °ËÃâ°á°ú
1¡¿104 ¼¼Æ÷·ÎºÎÅÍ ¾òÀº H3K4me3ÀÇ peak profileÀº 2.5¡¿105 ¼¼Æ÷ÀÇ °á°ú¿Í À¯»çÇÔÀ» È®ÀÎÇÏ¿´´Ù.

¡ß Á¦Ç° »ç¿ëÀ» °ËÅä ÁßÀÎ ºÐ²² »ç¿ëÀÚÀÇ ÇѸ¶µð
SMARTer¢ç ThruPLEX¢ç DNA-seq KitÀ» ÀÌ¿ëÇÏ¸é ¼Ò·®ÀÇ DNA·Îµµ ChIP-seq ºÐ¼®À» ÇÒ ¼ö ÀÖ½À´Ï´Ù. ¹°·Ð, »ùÇ÷®ÀÌ ¼Ò·®ÀÎÁö ¾Æ´ÑÁö¸¦ È®ÀÎÇÏ´Â °ÍÀÌ ¾î·Á¿î ºÐ¿¡°Ôµµ ¸Å¿ì ÃßõÇÕ´Ï´Ù. °£ÆíÇÏ°í È¿À²ÀûÀ¸·Î ChIP DNAÀÇ NGS Library¸¦ Á¦ÀÛÇÒ ¼ö Àֱ⠶§¹®¿¡, óÀ½ ½ÇÇèÇÏ´Â ºÐ²²µµ ¸Å¿ì ÃßõÇÒ ¸¸ÇÑ Á¦Ç°ÀÔ´Ï´Ù. (Prof. Shirahige Katsuhiko, The University of Tokyo)