- pg ¼öÁØ ±Ø¼Ò·® »ùÇÿ¡ ÃÖÀû - human, mouse, rat À¯·¡ÀÇ 250 pg - 100 ng total RNA ȤÀº 10 - 1,000°³ÀÇ intact cell
- UMI Àû¿ëÀ¸·Î ºÐ¼® Á¤È®µµ Áõ°¡ - PCR bias, sequencing error µîÀ» º¸Á¤ÇÏ¿© true variant¿Í rare mutationÀÇ Á¤È®ÇÑ ºÐ¼® °¡´É
- ´Ù¾çÇÑ »ùÇà Àû¿ë - FFPE, LCM, cell-free RNA µî ³·Àº qualityÀÇ RNA¿¡¼µµ ÀçÇö¼º ³ôÀº °á°ú È®ÀÎ
- ºü¸£°í °£ÆíÇÑ ÇÁ·ÎÅäÄÝ - Àü °úÁ¤ 6.5½Ã°£¸¸¿¡ ¿Ï·á, buffer Á¶¼ºÀÇ ¾÷±×·¹À̵å·Î library purification ½Ã°£ ´ÜÃà
- Illumina¢ç Ç÷§Æû¿¡ ȣȯ - Illumina¢ç ºÐ¼®¿ë index¸¦ Àû¿ëÇÏ¿© ÃÖ´ë 384°³ pooling °¡´É
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SMART-Seq¢ç Total RNA Pico Input with UMIs (ZapR¢â Mammalian)Àº picogram input (250 pg - 10 ng total RNA, 10 - 1,000 intact cell)ÀÇ ´Ù¾çÇÑ RNA (high quality, partially degraded, low quality total RNA)¿¡¼ È¿°úÀûÀ¸·Î Illumina¢ç sequencing library¸¦ Á¦ÀÛÇϱâ À§ÇØ °³¹ßµÇ¾ú´Ù. ±Ø¼Ò·®ÀÇ total RNA»Ó ¾Æ´Ï¶ó FFPE, LCM, cell-free RNA µî RNAÀÇ integrity°¡ ÇöÀúÈ÷ ³·Àº »ùÇÿ¡¼µµ NGS library Á¦ÀÛÀÌ °¡´ÉÇÏ¸ç ºü¸£°í °£ÆíÇÑ ÇÁ·ÎÅäÄÝÀÌ Æ¯Â¡À¸·Î RT¿Í PCR ´Ü°è¿¡¼ index¿Í adapter¸¦ Ãß°¡ÇØ strand Á¤º¸ (sense/antisense)¸¦ º¸À¯ÇÏ´Â library¸¦ Á¦ÀÛÇÒ ¼ö ÀÖ´Â Á¡ÀÌ Æ¯Â¡ÀÌ´Ù. ¶ÇÇÑ RT °úÁ¤¿¡¼ 8 nucleotide (nt)ÀÇ unique molecular identifier (UMI) ¼¿À» Ãß°¡ÇÏ¿© PCR bias¿Í sequencing error¸¦ º¸Á¤ÇÒ ¼ö ÀÖ¾î true variant¿Í rare mutationÀÇ Á¤È®ÇÑ ºÐ¼®ÀÌ °¡´ÉÇÏ´Ù.
ÀÌ Á¦Ç°Àº ´ç»çÀÇ µ¶ÀÚÀûÀÎ SMART (Switching Mechanism at 5¡¯ end of RNA Template) ±â¼ú¿¡ stranded RNA-seqÀ» À§ÇÑ °³¼±»çÇ×À» ´õÇØ ÇÁ·ÎÅäÄÝÀ» °£ÆíÈÇÏ°í sequencing È¿À²À» ³ô¿´´Ù. À̸¦ ÅëÇØ high- ¶Ç´Â low-qualityÀÇ total RNA ¸ðµÎ¸¦ ºÐ¼®ÇÒ ¼ö ÀÖ°í, strand Á¤º¸¸¦ °¡Áø sequencing library¸¦ Á¦ÀÛÇÒ ¼ö ÀÖ´Ù. ¶ÇÇÑ ZapR ±â¼úÀ» Àû¿ëÇÏ¿© rRNAÀ¯·¡ ¼¿À» cDNA ÇÕ¼º ÈÄ¿¡ Á¦°ÅÇÒ ¼ö ÀÖÀ½À¸·Î total RNA¸¦ ¹Ù·Î »ç¿ëÇÒ ¼ö ÀÖ¾î ±Ø¼Ò·®ÀÇ RNA ºÐ¼®¿¡ ¸Å¿ì ÀûÇÕÇÏ´Ù.
*±× ¿Ü »ùÇà input ¾ç¿¡ µû¸¥ Ãßõ Á¦Ç°:
- 10 ng - 100 ng total RNA (rRNA Á¦°Å ¹ÌÆ÷ÇÔ) : SMART-Seq¢ç Total RNA Mid Input (Code 635049)
- Mammalian total RNA 100 ng-1 ¥ìg (rRNA Á¦°Å Æ÷ÇÔ) : SMART-Seq¢ç Total RNA High Input (RiboGone¢â Mammalian) (Code 635046)
±×¸² 1. SMART-Seq¢ç Total RNA Pico Input with UMIs (ZapR¢â Mammalian) ÇÁ·Î¼¼½º.
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- Strand Á¤º¸¸¦ °¡Áø NGS library Á¦ÀÛ
- Illumina¢ç Ç÷§Æû RNA-seq
- ´Ù¾çÇÑ quality¿¡ total mammalian RNA¿¡¼ coding ¹× non-coding Á¤º¸ È®ÀÎ
(FFPE »ùÇÿ¡¼ Á¤Á¦ÇÑ RNA »ç¿ë °¡´É)
ÀÚ¼¼ÇÑ ±¸¼ºÀº CoA¸¦ Âü°íÇϼ¼¿ä.
RNA-Seq ·¯´×¼¾ÅÍ¿¡¼ ´õ ¸¹Àº µ¥ÀÌÅÍ¿Í FAQ¸¦ È®ÀÎÇÏ½Ç ¼ö ÀÖ½À´Ï´Ù.