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Home > ÀüÁ¦Ç°º¸±â > NGS °ü·Ã > Immune-Seq > [New]UMI Àû¿ë Human BCR profiling(v2)
RACE±â¼úÀ» È°¿ëÇÑ °í°¨µµ BCR repertoire profiling ºÐ¼®

[New]UMI Àû¿ë Human BCR profiling(v2)

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(ºÎ°¡¼¼º°µµ)
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Clontech
634777
SMART-Seq¢ç Human BCR (with UMIs)
°ü·ÃÇмú ±¸¸ÅÇϱâ
24ȸ
(Package)
3,234,000¿ø  Á¦Á¶»ç ÆäÀÌÁö·Î ¹Ù·Î°¡±â x341238
Clontech
634778
SMART-Seq¢ç Human BCR (with UMIs)
°ü·ÃÇмú ±¸¸ÅÇϱâ
96ȸ
(Package)
10,792,000¿ø  Á¦Á¶»ç ÆäÀÌÁö·Î ¹Ù·Î°¡±â
Clontech
634776
SMART-Seq¢ç Human BCR (with UMIs)
°ü·ÃÇмú ±¸¸ÅÇϱâ
384ȸ (634778 x 4)
38,852,000¿ø  Á¦Á¶»ç ÆäÀÌÁö·Î ¹Ù·Î°¡±â

¡Ø º¯°æ»çÇ×

º» Á¦Ç°Àº ±âÁ¸ SMARTer® Human BCR IgG IgM H/K/L Profiling KitÀÇ ¾÷±×·¹À̵å Á¦Ç°ÀÔ´Ï´Ù. Á¦Ç°»ç¿ë À¯¿¬¼ºÀ» À§ÇØ index°¡ kit¿¡ Æ÷ÇԵǾî ÀÖÁö ¾ÊÀ¸¸ç, º°µµ·Î ±¸¸Å°¡ ÇÊ¿äÇÕ´Ï´Ù.
>> ȣȯµÇ´Â Unique Dual Index kits

ÀÚ¼¼ÇÑ ³»¿ëÀº ´ÙÀ½ ¾È³»ÆäÀÌÁö¸¦ Âü°íÇϼ¼¿ä! Á¦Ç° ¼±ÅÃÀÌ ½¬¿öÁý´Ï´Ù.
>> RNA-Seq Á¦Ç°¸íÀÌ ÀÌ·¸°Ô ¹Ù²ò´Ï´Ù
  • Complete BCR repertoire profiling : SMART ±â¼ú°ú 5’ RACE¹ýÀ» Á¢¸ñ½ÃÄÑ all human BCR heavy chain (IgG, IgM, IgA, IgD, IgE)°ú light chain (IgK, IgL)À» °í°¨µµ·Î °ËÃâÇÒ ¼ö ÀÖ´Ù.
  • Unique Dual Index Kit (º°¸Å)¿Í ȣȯ : ¸ñÀû¿¡ µû¶ó multiplexingÀÌ °¡´ÉÇϸç, ÃÖ´ë 384°³ÀÇ »ùÇÃÀ» Çѹø¿¡ ºÐ¼®ÇÒ ¼ö ÀÖ´Ù. UDI¸¦ Àû¿ëÇÏ¿© high-throughput±â±â¿¡¼­ index hoppingÀ» ³·Ãâ ¼ö ÀÖ´Ù.
  • ³Ð¾îÁø input range : PBMCsÀÇ high-integrity RNA (10 ng–1 §¶), B cells (1–100 ng), spleen ¶Ç´Â bone marrow (10 ng), whole blood (100 ng) ¸ðµÎ Àû¿ë °¡´É
  • Á¤È®µµ Çâ»ó : UMI ±â¹ÝÀ¸·Î PCR duplicates, errors º¸Á¤
  • °£´ÜÇÑ ¿öÅ©Ç÷οì : heavy, light immunoglobulin chain¿¡ ´ëÇÑ primer pool°ú enzyme premix Á¦°ø
  • High-throughput workflow¸¦ À§ÇÑ automation system Àû¿ë°ú miniaturization °¡´É
Á¦Ç°¼³¸í
SMART-Seq® Human BCR (with UMIs)Àº 5’ RACE±â¼ú (Rapid Amplification of cDNA Ends)°ú NGS ºÐ¼® ±â¹ýÀ» Á¶ÇÕÇÑ Á¦Ç°À¸·Î, RNA »ùÇ÷κÎÅÍ ¾ÆÁÖ ¹Î°¨ÇÏ°í Á¤È®ÇÑ BCR profilingÀ» À§ÇÑ ÃÖÀûÈ­µÈ ½Ã½ºÅÛÀ» Á¦°øÇÑ´Ù.
SMART (Switching Mechanismat 5' end of RNA Template) full-length cDNAÇÕ¼º±â¼úÀ» 5’RACE¹ý°ú Á¢¸ñ½ÃÄÑ B-cell receptor (BCR) heavy chain°ú light chain (IgK/L)ÀÇ V(D)J variable regionÀ» ¿ÏÀüÇÏ°Ô Ä¸Ã³ÇÑ´Ù.
º» Á¦Ç°Àº peripheral blood mononuclear cells (PBMCs)¿¡¼­ ÃßÃâÇÑ 10 ng - 1 §¶ total RNA ¶Ç´Â B cell·ÎºÎÅÍ ÃßÃâÇÑ 1 - 100 ng total RNA·ÎºÎÅÍ °íÇ°ÁúÀÇ Illumina® ready library¸¦ Á¦ÀÛÇÒ ¼ö ÀÖÀ¸¸ç, Human immunoglobulin IgG, IgM, IgA, IgD, IgE, IgK, IgLÀ» °í°¨µµ·Î °ËÃâÇÒ ¼ö ÀÖ´Ù.
Á¦Ç° ³»¿¡´Â Unique Molecular Identifiers (UMIs)¸¦ Æ÷ÇÔÇÏ°í ÀÖ¾î, PCR ȤÀº sequencing error·ÎºÎÅÍ À¯·¡ÇÑ reads¸¦ Á¦°ÅÇÑ ÈÄ ºÐ¼®ÇÔÀ¸·Î½á º¸´Ù Á¤È®ÇÏ°í ½Å·Úµµ ÀÖ´Â °á°ú¸¦ ¾òÀ» ¼ö ÀÖ´Ù. ¶ÇÇÑ ÃÖ´ë 384°³ÀÇ multiplex Illumina library prepÀÌ °¡´ÉÇÑ UDI (Unique Dual Index Kit : º°µµ ÆǸÅ)¸¦ »ç¿ëÇϱ⿡ index hopingÀ¸·Î ÀÎÇÑ ´Ù¸¥ library »ùÇà À¯·¡ÀÇreads ¿À¿°À» ¹æÁöÇÒ ¼ö ÀÖ´Ù.


±×¸² 1. SMART-Seq® Human BCR (with UMIs)ÀÇ workflow
SMARTScribe Reverse Transcriptase¸¦ ÀÌ¿ëÇÏ¿© 1st strand cDNA¸¦ ÇÕ¼ºÇϸç mRNA 5'¸»´Ü¿¡ À̸£¸é template¿¡¼­ À¯·¡ÇÏÁö ¾ÊÀº nucleotide (3'-XXXX-5')°¡ ºÎ°¡µÈ´Ù. ÀÌ·¸°Ô ºÎ°¡µÈ non-templated nucleotide¿¡ SMART UMI Oligonucleotide°¡ °áÇÕÇÏ°í, ÀÌÈÄ ¿ªÀü»ç ¹ÝÀÀ¿¡ ÀÇÇØ SMART UMI Oligonucleotide ¼­¿­¿¡ »óº¸ÀûÀÎ nucleotide°¡ Ãß°¡·Î ÇÕ¼ºµÈ´Ù (Template-switching step). ÀÌ °úÁ¤¿¡¼­ ÇÕ¼ºµÈ 1st strand cDNA´Â µÎ ´Ü°èÀÇ gene specific PCR amplification °úÁ¤ (Nested PCR)À» °ÅÄ¡°Ô µÈ´Ù. Nested PCRÀ» °ÅÄ£ PCR »ê¹°Àº ´ëºÎºÐÀÇ reads°¡ BCR transcriptome¿¡ mappingµÈ´Ù.


±×¸² 2. ´Ù¾çÇÑ RNA input ¾ç¿¡¼­ ³ôÀº °¨µµ ¹× ÀçÇö¼ºÀ¸·Î clonotype °ËÃâ
SMART-Seq® Human BCR (with UMIs)À» »ç¿ëÇÏ¿©total human PBMC RNA 10, 100, 1,000 ngÀ¸·ÎºÎÅÍ Human BCR heavy chain (IgA/D/E/G/M) ¹× light chain (IgK/L) libraries¸¦ ¸¸µé¾ú´Ù.
Sequence read¸¦ Cogent NGS Immune Profiler Software¸¦ »ç¿ëÇÏ¿© ºÐ¼®ÇÑ °á°ú, »ùÇà ¾ç¿¡ °ü°è¾øÀÌ ¾ÈÁ¤ÀûÀÎ °á°ú°¡ ¾ò¾îÁ³´Ù.
±¸¼ºÇ° (ÀÚ¼¼ÇÑ ³»¿ëÀº CoA¸¦ ÂüÁ¶Çϼ¼¿ä)
Box 1:
  • Control Total RNA (1 §¶/§¡) ¡Ø
  • SMART UMI Oligo

Box 2:
  • dT Primer
  • First-Strand Buffer (5X)
  • SMARTScribeTM Reverse Transcriptase (100 U/§¡)
  • Nuclease-Free Water
  • BCR Enhancer
  • RNase Inhibitor (40 U/§¡)
  • Elution Buffer (10 mM Tris HCl, pH 8.5)
  • hBCR PCR1 Universal Forward
  • hBCR PCR Reverse
  • hBCR PCR2 HC Reverse
  • hBCR PCR2 LC Reverse
  • PrimeSTAR® GXL Premix

¡Ø Control RNA is human spleen Total RNA.
º¸Á¸
Box 1 : -70¡É
Box 2 : -20¡É
       BCR Enhancer, Nuclease-Free Water: -20¡É º¸°ü, À¶ÇØ ÈÄ¿¡´Â 4¡É º¸°ü
       Elution Buffer: -20¡É º¸°ü, À¶ÇØ ÈÄ¿¡´Â ½Ç¿Â º¸°ü
       ±× ¿Ü : -20¡É
°ü·Ã Á¦Ç°
- Multiplexed sequencing libraries Á¦ÀÛÀ» À§ÇÑ Unique Dual Index Kit (Code 634756 ¿Ü)
- TCR repertoire¸¦ Ź¿ùÇÑ °¨µµ·Î ºÐ¼® SMART-Seq ® Human TCR (with UMIs) (Code 634780 ¿Ü)
- Á¤È®µµ ³ôÀºPCR ¹ÝÀÀÀ» À§ÇÑ 2X premix PrimeSTAR® GXL Premix (Code R051A ¿Ü)
- DNA purification & size selectionÀ» À§ÇÑ SMARTer®-Seq™ Magnetic Separator-PCR Strip (Code 635011)

Keyword :

Immune-Seq
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