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Home > ÀüÁ¦Ç°º¸±â > NGS °ü·Ã > mRNA-Seq (Single cell) > High throughput, Automation mRNA-Seq Kit
SSv4 ±â¹Ý, High-throughput mRNA-seq ºÐ¼®

High throughput, Automation mRNA-Seq Kit

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(ºÎ°¡¼¼º°µµ)
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Clontech
634439
SMART-Seq¢ç HT Lysis Components
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96ȸ
(Package)
195,000¿ø  Á¦Á¶»ç ÆäÀÌÁö·Î ¹Ù·Î°¡±â x98121




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º» Á¦Ç°Àº SMART-Seq¢ç mRNA HT (Code 634791, 634795, 634796)°ú SMART-Seq¢ç mRNA HT LP (Code 634792, 634793, 634794) Á¦Ç°À¸·Î ¸®´º¾ó µÇ¾ú½À´Ï´Ù.
ÀÚ¼¼ÇÑ ³»¿ëÀº ´ÙÄ«¶óÄÚ¸®¾Æ (02-2081-2510)·Î ¹®ÀÇ ÁÖ¼¼¿ä.

* PLUS kit ³»¿¡´Â »ùÇ÷κÎÅÍ lllumina¢ç Ç÷§ÆûÀÇ ¶óÀ̺귯¸® Á¦ÀÛÀ» À§ÇÑ, library preparation kit¿Í single-use unique dual index (UDI)¸¦ Æ÷ÇÔÇÏ°í ÀÖ½À´Ï´Ù.

  • SSv4¸¦ ±â¹ÝÀ¸·Î, ´Ù·®ÀÇ »ùÇà ºÐ¼® ¹× ÀÚµ¿È­ Àû¿ë¿¡ ÃÖÀûÈ­
  • Easily automate the streamlined workflow - ÀûÀº ½Ã°£À¸·Î ´õ ¸¹Àº »ùÇà ºÐ¼® °¡´É
  • Unparalleled sensitivity - Single cell ¶Ç´Â 10 pg total RNA¿¡¼­ ºÐ¼® °¡´É (Input range: 1 - 100 cells or 10 pg - 1 ng of total RNA)
  • Single-tube workflows - cDNA ÇÕ¼ººÎÅÍ library °úÁ¤À» °¢°¢ single tube¿¡¼­ ÁøÇàÇÒ ¼ö ÀÖ¾î, »ùÇà ¼Õ½ÇÀ̳ª ¿À¿° µî ½ÇÇè °úÁ¤¿¡¼­ ¹ß»ýÇÒ ¼ö ÀÖ´Â ¿À·ù ÃÖ¼ÒÈ­
  • Increase your sequencing power - ¸¹Àº »ùÇÃÀ» ºÐ¼®ÇÒ ¼ö ÀÖ´Â Unique dual index (UDI) »ç¿ë
  • High library yield - ³ôÀº yieldÀÇ library¸¦ Á¦ÀÛÇÒ ¼ö ÀÖ¾î, NovaSeq¢â system°ú °°Àº high-throughput sequencer¿¡ Àû¿ëÇϰųª, ¿©·¯ Â÷·Ê ºÐ¼® °¡´É
Á¦Ç°¼³¸í
SMART-Seq¢ç HT Kit (SSv4-HT)¿Í SMART-Seq¢ç HT PLUS kit (SSv4-HT PLUS)´Â ±Ø¼Ò·®ÀÇ ¼¼Æ÷³ª RNA »ùÇÃÀ» ´ë·®(High-throughput)À¸·Î ºÐ¼®ÇÒ ¼ö ÀÖ´Â Á¦Ç°ÀÌ´Ù. SSv4-HT PLUS Á¦Ç°À» ÀÌ¿ëÇϸé, 10 pg - 1ng total RNA ¶Ç´Â 1 - 100°³ÀÇ ¼¼Æ÷¿¡¼­ RNA ÃßÃâ °úÁ¤ ¾øÀÌ, oligo(dT) primer¸¦ ÀÌ¿ëÇÑ °íÇ°Áú full-length cDNA¸¦ Á¦Á¶ÇÑ ÈÄ, lllumina¢ç Ç÷§ÆûÀÇ ¶óÀ̺귯¸® Á¦ÀÛ±îÁö ÁøÇàÇÒ ¼ö ÀÖ´Ù.
º» Á¦Ç°Àº SMART (Switching Mechanism at 5' End of RNA Template) ±â¼ú¿¡ Locked Nucleic Acid (LNA) ±â¼úÀ» Á¢¸ñÇÔÀ¸·Î½á template switchingÀÇ È¿À²À» ´õ¿í Çâ»ó½ÃÄ×´Ù. µû¶ó¼­, ÀÌÀü ¼¼´ëÀÇ Á¦Ç°º¸´Ù ´õ¿í ¸¹Àº ¼öÀÇ À¯ÀüÀÚ¼ö¸¦ °ËÃâÇÒ ¼ö ÀÖ¾î Àü¹ÝÀûÀÎ Àü»çü ¿¬±¸ (full transcriptome analysis)¿¡ ÀÖ¾î ¸Å¿ì À¯¿ëÇÏ´Ù. ¶ÇÇÑ, SMART-Seq¢ç v4 Ultra¢ç Low Input RNA Kit for Sequencing (Code 634888)ÀÇ ±â¼úÀ» ±â¹ÝÀ¸·Î ÇÏ°í ÀÖ¾î µ¿ÀÏÇÑ ¼º´ÉÀÇ ¹Î°¨µµ (sensitivity)¿Í ÀçÇö¼º (reproducibility)¸¦ º¸ÀÏ »Ó ¾Æ´Ï¶ó, FACS sorting ÀÌÈÄ ½ÇÇè ´Ü°è¸¦ ÃÖÀûÈ­ÇÏ¿©, hands-on timeÀ» °¨¼Ò½ÃÅ´À¸·Î½á Æí¸®¼ºÀ» ³ô¿´´Ù.
SMART-Seq¢ç HT PLUS kit (SSv4-HT 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·Î Á¦ÀÛÇÒ ¼ö ÀÖ´Ù. µû¶ó¼­ »ùÇÃÀÇ 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±â¼úÀ» ÀÌ¿ëÇÑ cDNA ÇÕ¼º °úÁ¤


±×¸² 2. Comparison of the SMART-Seq v4 and SMART-Seq HT kit workflows
(¿ÞÂÊ) SMART-Seq¢ç v4 Ultra¢ç Low Input RNA Kit for Sequencing (Code 634888)
(¿À¸¥ÂÊ) SMART-Seq¢ç HT Kit (Code 634437) (¿À¸¥ÂÊ)Àº ±âÁ¸ÀÇ SMART-Seq¢ç v4 (¿ÞÂÊ)ÀÇ ÇÁ·ÎÅäÄÝÀ» º¯ÇüÇÔÀ¸·Î½á, º¸´Ù °£ÆíÇÏ°í ºü¸£°Ô High-throughputÀ¸·Î NGS Library¸¦ Á¦ÀÛÇÒ ¼ö ÀÖ´Ù. FACS sortingÀ¸·Î Single cell ºÐ¸® ÈÄ, SMART-Seq¢ç v4ÀÇ °æ¿ì´Â ÃÑ 6¹øÀÇ Hands-on stepÀÌ ÇÊ¿äÇÑ µ¥ ¹ÝÇÏ¿©, SMART-Seq¢ç HT KitÀÇ °æ¿ì´Â ´Ü 3¹øÀÇ Hands-on stepÀ¸·Î cDNA ÇÕ¼ºÀ» ¿Ï·áÇÒ ¼ö ÀÖ´Ù.


±×¸² 3. FACS·Î ºÐ·ùÇÑ 293T cellsÀ» SMART-seq¢ç v4°ú SMART-Seq¢ç HT kits¸¦ ÀÌ¿ëÇØ ºÐ¼®ÇßÀ» ¶§, ³ôÀº ÀçÇö¼º°ú À¯ÀüÀÚ ¹ßÇö È®ÀÎ

Libraries generated from twenty-one individual 293T cells (table, top panel) were further analyzed to evaluate the reproducibility of gene expression measurements obtained for each cell with the SMART-Seq¢ç v4 kit (SSv4_1 to SSv4_12) and the SMART-Seq¢ç HT Kit (HT_1 to HT_9) (lower panel). The hierarchical clustering heat map shows Euclidean distances between all the cells and reports Pearson correlations ranging from 0.74 to 0.97. While the best correlations are observed between cells prepared with one or the other kit, the correlations are still very high between the two kits and the cells are not clustering based on the library preparation method. These data demonstrate that the modified workflow in the SMART-Seq¢ç HT Kit does not introduce major bias in measurement of gene expression levels.


±×¸² 4. SMART-Seq v4¿Í SMART-Seq HT kit·Î ºÐ¼®ÇßÀ» ¶§ÀÇ À¯ÀüÀÚ GC content ¹ßÇö ºñ±³

The libraries made from 10 pg of Mouse Brain Total RNA shown in the table (Figure 2) were further analyzed for GC content representation (see table, top panel). Genes were binned by GC content, and correlation plots were used to visualize the reproducibility of the expression levels (FPKM) of genes in each bin. The average gene counts are very reproducible for replicate samples analyzed using the SMART-Seq¢ç v4 (Panel A) or SMART-Seq¢ç HT kits (Panel B). Genes with high or low GC content (shown in red and blue, respectively) show similar expression levels in the SMART-Seq¢ç v4 and SMART-Seq¢ç HT kits (Panel C). Thus, the One-Step RT PCR reaction introduced in the new SMART-seq¢ç HT Kit maintains the representation of the low- and high-GC content genes


±×¸² 5. High overlap of transcripts identified with the SMART-Seq v4 and SMART-Seq¢ç HT kits
Libraries prepared from 10 pg of Mouse Brain Total RNA shown in Table 1 were further evaluated for the overlap in the number of transcripts identified (FPKM >0.1) between technical replicates within each kit, and found to be very similar (61-63% overlap) (Panel A). Transcripts identified by all three replicates for each kit were then compared against each other, indicating an overlap of 71%. The overlapping transcripts have an average expression level of 37 FPKM, while the transcripts uniquely identified with individual kits are less abundant, averaging between 6-7 FPKM, indicating that the transcripts more likely to not be identified are the ones expressed at a low level.


±×¸² 6. SSv4-HT PLUS¸¦ ÀÌ¿ëÇÑ library¿¡¼­ Nextera¢ç XTº¸´Ù ´õ ³ôÀº yield¿Í À¯ÀüÀÚ °ËÃâ

(Panel A) SMART-Seq¢ç HT was used to produce cDNA from 10 pg of Mouse Brain Total RNA in triplicate. Illumina¢ç-compatible libraries were generated using SMART-Seq¢ç PLUS or Nextera¢ç XT (Illumina¢ç) library prep kits, and sequenced on a NextSeq 500 system. The reads were normalized to 4M paired-end reads and analyzed as described in the technical note.
(Panel B) Library yields obtained with SMART-Seq¢ç Library Prep Kit are higher than those generated with Nextera XT.
(Panel C) The SMART-Seq Library Prep Kit identified 10% more genes than Nextera¢ç XT.
Àû¿ë
±Ø¼Ò·® »ùÇ÷κÎÅÍ 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¢ç Library Prep kit (SSv4-HT 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.

Keyword : Single cell,´ÜÀϼ¼Æ÷,½Ì±Û¼¿,single cell analysis,single cell NGS,full length,Ultra low input,low input,±Ø¼Ò·®,automation,mRNA,mRNA-seq,cDNA,cDNA-seq,NGS,Next Generation Sequencing,SMART,,smart-seq,Rubicon,·çºñÄÜ

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