Accelerating chromatin mapping with single-cell ATAC-seq
Epigenomic Á¤º¸´Â À¯ÀüÀÚ Á¶Àý°ú °ü·ÃÇÏ¿© ¸¹Àº Á¤º¸¸¦ Á¦°øÇÏÁö¸¸, µ¿ÀÏÇÑ À¯ÇüÀÇ ´ÜÀÏ ¼¼Æ÷¶óµµ µ¶Æ¯ÇÑ epigenomic profileÀ» °¡Áú ¼ö Àֱ⠶§¹®¿¡ ¸Å¿ì º¹ÀâÇÏ´Ù. ÃÖ±Ù ¸î ³â µ¿¾È single cell NGS ¹æ¹ýÀÌ ¸¹ÀÌ ¹ßÀüÇßÁö¸¸
Single cell¿¡¼ÀÇ chromatin ºÐ¼®Àº º¹ÀâÇÏ°í ¸Å¿ì ÀûÀº ¾çÀ» ÀÌ¿ëÇÏ¿© ºÐ¼®ÇØ¾ß Çϸç, ºñ¿ë ¶ÇÇÑ ÀûÁö ¾Ê´Ù. ÃÖ±Ù Stanford UniversityÀÇ Greenleaf ¿¬±¸½ÇÀº
ICELL8¢ç Single-Cell SystemÀ» »ç¿ëÇÏ¿© single cell·ÎºÎÅÍ sequencing library¸¦ preparationÇÏ´Â ¸Å¿ì °£´ÜÇÏ°í ½Å¼ÓÇÑ ATAC-seq workflow¸¦ Takara Bio USA, Inc.(TBUSA)¿Í °øµ¿À¸·Î °³¹ßÇÏ¿´´Ù
(
Ŭ¸¯). ÀÌ ¹æ¹ýÀ» ÀÌ¿ëÇϸé Chip»ó¿¡¼ 4~5½Ã°£ ¸¸¿¡ 1,000°³ ÀÌ»óÀÇ single cell¿¡¼ library¸¦ Á¦ÀÛÇÒ ¼ö ÀÖ´Ù.
Chromatin accessibility: a sequence of challenges
ChromatinÀº À¯ÀüÀÚ ¹ßÇöÀ» Á¶ÀýÇÏ´Â µ¥ Áß¿äÇÑ ¿ªÇÒÀ» Çϸç, ChromatinÀÇ º¯ÇüÀ» ÅëÇØ ´Ü¹éÁú ¹× Àü»ç ÀÎÀÚÀÇ DNA °áÇÕ·ÂÀ» º¯È½ÃÄÑ À¯ÀüÀÚ Àü»ç¸¦ ÃËÁøÇϰųª ¾ïÁ¦ÇÒ ¼ö ÀÖ´Ù. ÇÏÁö¸¸, ±âÁ¸ÀÇ Chromatin mapping ±â¼úÀº ³ôÀº ºñ¿ë¿¡µµ ºÒ±¸ÇÏ°í ºÐ¼®·®ÀÌ Á¦ÇÑÀûÀÌ°í (low-throughput), ½ÇÇè °úÁ¤ÀÌ ¸Å¿ì º¹ÀâÇÏ´Ù´Â ÇÑ°è°¡ ÀÖ¾ú´Ù. ¶ÇÇÑ µ¿ÀÏÇÑ Á¶Á÷À̳ª ¼¼Æ÷ À¯Çü¿¡¼µµ ´ë·® ºÐ¼®À» ÁøÇàÇßÀ» ¶§ Single cell¿¡¼ º¸ÀÌ´Â µ¶Æ¯ÇÑ chromatin ±¸Á¶¸¦ ¹ß°ßÇÏÁö ¸øÇÏ´Â °æ¿ìµµ Á¾Á¾ ¹ß»ýÇÒ ¼ö ÀÖ´Ù.
A singular protocol: the new approach to map chromatin
ÀÌ·¯ÇÑ ÇѰ踦 ±Øº¹Çϱâ À§ÇØ, Greenleaf ¿¬±¸¼Ò¿¡¼´Â ´ÙÄ«¶ó¹ÙÀÌ¿ÀÀÇ
ICELL8¢ç Single-Cell System¸¦ ÀÌ¿ëÇØ
high-throughput scATAC-seq protocolÀ» °³¹ßÇÏ¿´´Ù (±×¸² 1). ICELL8 systemÀ» ÀÌ¿ëÇϸé single cellÀ» 5,184-nanowell blank chip (±×¸² 1)¿¡ ÀÚµ¿ ºÐÁÖÇÒ ¼ö ÀÖÀ¸¸ç, Hoechst¿Í propidium iodide ¿°»ö¹ýÀ» »ç¿ëÇÏ¿© »ì¾ÆÀÖ´Â single cellÀÌ µé¾îÀÖ´Â wellÀ» ÀÚµ¿À¸·Î ½Äº°ÇÒ ¼ö ÀÖÀ¸¹Ç·Î ¸ñÀûÇÏ´Â well¿¡¸¸ ½Ã¾àÀ» ºÐÁÖÇÒ ¼ö ÀÖ´Ù. Chip»ó¿¡¼ tagmentation, index Ãß°¡ ¹× PCR amplificationÀÌ (±×¸² 1)¿¡¼ ÁøÇàµÇ¹Ç·Î ±â¼úÀû °¡º¯¼ºÀ» ÃÖ¼ÒÈÇÏ°í 4-5½Ã°£ÀÇ °£´ÜÇÑ workflow·Î ½ÇÇèÀÌ °¡´ÉÇÏ´Ù. ±×·± ´ÙÀ½ PCR ampliconÀ» chip¿¡¼ ȸ¼ö, poolingÇÏ¿©, size selection bead·Î Á¤Á¦ÇÏ¿© sequencing library¸¦ Á¦ÀÛÇÒ ¼ö ÀÖ´Ù.
±×¸² 1. ICELL8 Single-cell systemÀ» ÀÌ¿ëÇÑ ATAC-seqÀÇ ½ÇÇè °úÁ¤
Cells are automatically dispensed, imaged, and live single-cell wells are selected
(Panel 1). A transposition mix containing Tn5 transposase is added to selected wells and subjected to thermal cycling
(Panel 2).
Index 1 is added in a high-EDTA mix to remove bound transposase, followed by the addition of index 2 in a high-MgCl2 mix to quench the EDTA
(Panels 3-4). Selected wells are then subjected to on-chip PCR amplification
(Panel 5). Figure by Anja Mezger
et. al., used under CC BY 4.0.
It pays to innovate: chromatin profiles reveal distinct cell types
¸»ÃÊÇ÷¾× ´ÜÇÙ¼¼Æ÷(PBMC)´Â ´ÜÇÙ±¸, B¼¼Æ÷, T¼¼Æ÷·Î ±¸¼ºµÇ¾î, Ç÷¾× ³» ´Ù¾çÇÑ ±â´ÉÀ» Áö´Ñ´Ù. Greenleaf ¿¬±¸ÆÀÀº ¼¼ ¸íÀÇ donor Ç÷¾×À¸·ÎºÎÅÍ single PBMC¸¦ ÃßÃâÇÑ µÚ, scATAC-seq ÇÁ·ÎÅäÄÝ¿¡ µû¶ó chromatin profilingÀ» ÁøÇàÇß´Ù. ³î¶ø°Ôµµ
´ÜÇÙ±¸, B¼¼Æ÷, T¼¼Æ÷¿¡¼ Àü»ç ½ÃÀÛ ºÎÀ§ ³»ÀÇ chromatin accessibility patternÀÌ ¶Ñ·ÇÇÏ°Ô ±¸ºÐµÇ¾úÀ¸¸ç (±×¸² 2), scATAC-seqÀ» ÀÌ¿ëÇÏ¸é º¹ÀâÇÑ »ùÇà ³»¿¡¼ ¼¼Æ÷ Á¾·ù¿¡ µû¸¥ ºÐ·ù°¡ °¡´ÉÇÔÀ» È®ÀÎÇÏ¿´´Ù.
±×¸² 2. PBMC·ÎºÎÅÍ scATAC-seq¸¦ ÁøÇàÇßÀ» ¶§ º¸ÀÌ´Â ¼¼Æ÷ ŸÀÔ º° chromatin accessibility patterns
Chromatin accessibility for all cells (Panel A), PU.1 (Panel B), C/EBP¥á (Panel C), and RUNX1 (Panel D) demonstrates discrete clusters of monocytes and B and T cells. Figure by
Anja Mezger et. al., used under CC BY 4.0.
[¿ø¹®] Accelerating chromatin mapping with single-cell ATAC-seq
[Âü°í¹®Çå]
- Mezger, A.
et al. High-throughput chromatin accessibility profiling at single-cell resolution.
Nat. Commun. 9, 3647 (2018).