* PLUS kit´Â »ùÇ÷κÎÅÍ lllumina¢ç ºÐ¼®¿ë library Á¦ÀÛÀ» À§ÇÑ, library preparation kit¿Í single-use unique dual index (UDI)¸¦ Æ÷ÇÔÇÏ°í ÀÖ½À´Ï´Ù.
- Robust, full-length chemistry - SMART-seq2¸¦ Æ÷ÇÔÇÑ ±âÁ¸ÀÇ ¸ðµç single cell full-length ºÐ¼®¹ýÀ» ¶Ù¾î³Ñ´Â ¼º´É
- Easy, plate-based workflow - FACS µîÀ¸·Î ºÐ¸®ÇÑ single cell¿¡ ¹Ù·Î Àû¿ë °¡´É
- Single-tube workflows - cDNA ÇÕ¼ººÎÅÍ library Á¦ÀÛ °úÁ¤À» °¢°¢ single tube¿¡¼ ÁøÇàÇÒ ¼ö ÀÖ¾î, »ùÇà ¼Õ½ÇÀ̳ª ¿À¿° µî ½ÇÇè °úÁ¤¿¡¼ ¹ß»ýÇÒ ¼ö ÀÖ´Â ¿À·ù ÃÖ¼ÒÈ
- Unparalleled sensitivity and reproducibility - ³ôÀº À¯ÀüÀÚ °ËÃâ È¿À²À» ÀÌ¿ëÇÑ ÃÖ°í ¼º´ÉÀÇ ´ÜÀÏ ¼¼Æ÷ RNA-seq (ƯÈ÷, RNA ÇÔ·®ÀÌ ¸Å¿ì ³·Àº ¼¼Æ÷ ¹× ÇÙ¿¡ Àû¿ë)
- High library yield - ³ôÀº yieldÀÇ library¸¦ Á¦ÀÛÇÒ ¼ö ÀÖ¾î, NovaSeq system°ú °°Àº high-throughput sequencer¿¡ Àû¿ëÇϰųª, ¿©·¯ Â÷·Ê ºÐ¼® °¡´É
- Highly scalable workflow - ´ë·® (High-throughput) ½ÇÇè Àû¿ë°¡´É, ÀÚµ¿È ±â±â Àû¿ë¿¡ ÃÖÀû
Á¦Ç°¼³¸í
SMART-Seq¢ç Single Cell Kit (SSsc)¿Í SMART-Seq¢ç Single Cell PLUS Kit (SSsc PLUS)´Â ³ôÀº ¹Î°¨µµ¿Í ÀçÇö¼ºÀ¸·Î single cell ȤÀº single nuclei¸¦ ºÐ¼®ÇÒ ¼ö ÀÖ´Ù. SSsc PLUS Á¦Ç°À» ÀÌ¿ëÇϸé, single cell¿¡¼ RNA¸¦ ÃßÃâÇÏÁö ¾Ê°í, ¹Ù·Î oligo(dT) primer¸¦ ÀÌ¿ëÇØ °íÇ°Áú full-length cDNA¸¦ Á¦Á¶ÇÑ ÈÄ, lllumina¢ç Ç÷§ÆûÀÇ library Á¦ÀÛ±îÁö ÁøÇàÇÒ ¼ö ÀÖ´Ù. Á¦Ç°À» ÀÌ¿ëÇÏ¿© 2 pg total RNA¿Í RNA ÇÔ·®ÀÌ ³·Àº °ÍÀ¸·Î ¾Ë·ÁÁ® ÀÖ´Â ¸»ÃÊÇ÷¾× ´ÜÇÙ¼¼Æ÷ (PBMCs)¿Í °°Àº single cell¿¡¼ ¿ì¼öÇÑ °á°ú¸¦ È®ÀÎÇÏ¿´´Ù.
º» Á¦Ç°Àº single cell ºÐ¼®À» À§ÇØ SMART (Switching Mechanism at 5' End of RNA Template) ±â¼úÀ» ¾÷±×·¹À̵åÇÏ¿© °³¹ßµÈ ½Ã½ºÅÛÀ¸·Î, ´õ ¸¹Àº read¿Í À¯ÀüÀÚ¸¦ °ËÃâÇÒ ¼ö ÀÖ´Ù. SSsc´Â ³ôÀº ÀçÇö¼º°ú gene-body coverage »Ó ¾Æ´Ï¶ó GC-rich Àü»çüµµ Á¤È®ÇÏ°Ô cDNA·Î ÇÕ¼ºÇÒ ¼ö ÀÖ´Ù.
SMART-Seq¢ç Single Cell PLUS Kit (SSsc PLUS)¿¡ Æ÷ÇԵǾî ÀÖ´Â library preparation kit´Â enzymatic fragmentation¿Í stem-loop adaptor¸¦ ÇÔ²² »ç¿ëÇÏ¿©, 2½Ã°£ ³» single tube¿¡¼ purification °úÁ¤ ¾øÀÌ cDNA·ÎºÎÅÍ library¸¦ Á¦ÀÛÇÒ ¼ö ÀÖ´Ù. µû¶ó¼, »ùÇà ¼Õ½ÇÀ̳ª ¿À¿° µî ½ÇÇè °úÁ¤¿¡¼ ¹ß»ýÇÒ ¼ö ÀÖ´Â ¿À·ù¸¦ ÃÖ¼ÒÈ ÇÒ ¼ö ÀÖ´Ù. ÀÌ °úÁ¤À» ÅëÇØ Á¦ÀÛµÈ library´Â SMART-Seq2 µî ±âÁ¸ÀÇ ¹æ¹ý ¹× ´ç»ç ±âÁ¸ Á¦Ç° (SMART-Seq¢ç v4)¿¡ ºñÇØ ÀçÇö¼ºÀÌ ³ô°í, ÈξÀ ¿ì¼öÇÒ »Ó ¾Æ´Ï¶ó, ³ôÀº yield·Î Á¦À۵Ǿî high-throughput sequencer¿¡ Àû¿ëÇϰųª, ¿©·¯ Â÷·Ê ºÐ¼®ÇÒ ¼ö ÀÖ´Ù.
±×¸² 1. SMART-Seq¢ç Single Cell Kit¿Í SMART-Seq2 ¹ý°úÀÇ °á°ú ºñ±³
SMART-Seq2À» ÀÌ¿ëÇßÀ» ¶§ º¸´Ù SMART-Seq¢ç Single Cell KitÀ» ÀÌ¿ëÇßÀ» ¶§, mitochondria À¯·¡ À¯ÀüÀڷκÎÅÍ ÈξÀ ´õ ³ôÀº mapped reads¿Í º¸´Ù ¸¹Àº À¯ÀüÀÚ°¡ °ËÃâµÊÀ» È®ÀÎÇÏ¿´´Ù.
Single cells from the lymphoblastoid cell line GM12878 were processed with the SMART-Seq¢ç Single Cell Kit (SSsc; 18 cells) or the Smart-seq2 method (Smart-seq2; 20 cells) using 19 cycles of PCR. RNA-seq libraries were generated and sequences analyzed (after normalizing all samples to 1.75 million paired-end reads).
(Panel A) The read distribution was different between the two chemistries, with a drastically higher number of reads mapping to the mitochondrial genome (and therefore, fewer reads available for gene identification) with Smart-seq2 chemistry.
(Panel B) More genes were detected in the cells processed with SSsc.
±×¸² 2. SMART-Seq¢ç Single Cell Kit¿Í ´ç»ç ±âÁ¸ Á¦Ç°ÀÎ SMART-Seq v4¿ÍÀÇ ºñ±³
´ç»ç ±âÁ¸ Á¦Ç°ÀÎ SMART-Seq¢ç v4À» ÀÌ¿ëÇßÀ» ¶§ º¸´Ù SMART-Seq¢ç Single Cell KitÀ» ÀÌ¿ëÇßÀ» ¶§,
´õ ³ôÀº cDNA yield¸¦ ¾ò¾ú°í, reads¿¡ °ü°è¾øÀÌ °ËÃâµÇ´Â À¯ÀüÀÚ ¼ö°¡ 60% ÀÌ»óÀÓÀ» È®ÀÎÇÏ¿´´Ù.
(Panel A) 12 single cells from lymphoblastoid cell line GM22601 were processed with SSv4 or SSsc using 19 cycles of PCR. All samples were normalized to 1.25 million paired-end reads. The cDNA yield generated with SSsc was drastically higher than that generated with SSv4.
(Panel B) About 50 single PBMCs from one donor were processed with SSv4 or SSsc. About 60% more genes were detected in the cells processed with SSsc, regardless of the number of reads used for the analysis.
±×¸² 3. SSsc PLUS¸¦ ÀÌ¿ëÇÑ library¿¡¼ Nextera¢ç XTº¸´Ù ´õ ³ôÀº yield¿Í À¯ÀüÀÚ °ËÃâ
(Panel A) The SMART-Seq¢ç Single Cell Kit was used to produce cDNA, in triplicate, from 10 pg of Mouse Brain Total RNA. Illumina¢ç-compatible libraries were then generated using either the SMART-Seq¢ç Library Prep Kit portion of the SSsc PLUS kit or Nextera¢ç XT. Libraries were sequenced on a NextSeq 500. The reads were normalized to 4M paired-end reads and analyzed as described in the technical note.
(Panel B) Library yields obtained with the SSsc PLUS kit are higher than yields produced with Nextera¢ç XT.
(Panel C) The SSsc PLUS kit also identified more genes compared to Nextera¢ç XT.
Àû¿ë
Single cell À¯·¡ full-length transcriptome ºÐ¼® (transcript isoforms, gene fusions, point mutations µî)À» À§ÇØ, cDNA ÇÕ¼º °úÁ¤¿¡¼ »ç¿ëÇÒ ¼ö ÀÖ´Ù. º» Á¦Ç°À» ÀÌ¿ëÇØ ÇÕ¼ºÇÑ cDNA´Â SMART-Seq Library Prep kit (SSsc PLUS kit ³» Æ÷ÇÔ, º°µµ ±¸¸Å ºÒ°¡) ¸¦ ÀÌ¿ëÇÏ¿© Illumina¢ç ºÐ¼®¿ë sequencing library·Î Á¦ÀÛÇÒ ¼ö ÀÖ´Ù.
ÀÚ¼¼ÇÑ ±¸¼ºÀº CoA¸¦ Âü°íÇϼ¼¿ä.
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