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NGS °ü·Ã
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Immune-Seq
> Human BCR IgG IgM H/K/L Profiling Kit(v1)
Human BCR V(D)J ¿µ¿ª ¼¿ ºÐ¼® (BCR profiling)
Human BCR IgG IgM H/K/L Profiling Kit(v1)
Á¦Á¶»ç
Á¦Ç°ÄÚµå
Á¦Ç°¸í
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°¡°Ý
(ºÎ°¡¼¼º°µµ)
ºñ°í
»ç¿ëÀڸŴº¾ó
Clontech
634466
SMARTer¢ç Human BCR IgG IgM H/K/L Profiling Kit
12ȸ
(Package)
2,346,000¿ø
Clontech
634467
SMARTer¢ç Human BCR IgG IgM H/K/L Profiling Kit
48ȸ
(Package)
6,809,000¿ø
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PGT-A
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[¼±Åð¡À̵å] Immune-se..
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[New]UMI Àû¿ë Human BC..
[New]UMI Àû¿ë Mouse TC..
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[±â¼ú°³¿ä] ThruPLEX & ..
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* SMARTer
®
Human BCR IgG IgM H/K/L Profiling Kit´Â
SMART-Seq
®
Human BCR (with UMIs)
·Î ¾÷±×·¹ÀÌµå µÇ¾ú½À´Ï´Ù.
Input sample amount
- Peripheral blood mononuclear cell (PBMC)·ÎºÎÅÍ ÃßÃâÇÑ 10 ng - 1 ¥ìg total RNA
- B cell·ÎºÎÅÍ ÃßÃâÇÑ 1 - 100 ng total RNA
PCR error, duplication error ¶Ç´Â sequencing error·ÎºÎÅÍ ÆÄ»ýµÈ reads Á¦°Å ¸ñÀûÀÇ UMI based correction
ÃÖÀûÈµÈ cDNA library Á¦ÀÛ¹ýÀ¸·Î ¾ÆÁÖ ¹Î°¨ÇÏ°í ƯÀÌÀûÀÎ BCR clonotype °ËÃâ
Human IgG subclass¸¸ ƯÀÌÀûÀ¸·Î Á¤È®ÇÏ°Ô ÁõÆøÇÏ¿© ¿°±â¼¿ ºÐ¼®
µ¿ÀÏ »ùÇÃÀÇ ´Ù¾çÇÑ »ç½½ (Chain; IgG, IgM, IgK, IgL)ÀÇ Á¤¹ÐÇÑ °ËÃâÀ» À§ÇØ ÃÖÀûÈµÈ PCR Pooling Strategy
½ÃÄö½Ì µ¥ÀÌÅÍ ºÐ¼®À» À§ÇÑ
Takara Bio Immune Profiler Software
Á¦°ø
Á¦Ç°¼³¸í
SMARTer
¢ç
Human BCR IgG IgM H/K/L Profiling KitÀº 5¡¯ RACE¹ý (Rapid Amplification of cDNA Ends)°ú NGS ±â¼úÀ» Á¶ÇÕÇÑ Á¦Ç°À¸·Î½á, RNA »ùÇ÷κÎÅÍ ¾ÆÁÖ ¹Î°¨ÇÏ°í Á¤È®ÇÑ BCR profilingÀ» À§ÇØ ÃÖÀûÈµÈ ÇÁ·ÎÅäÄÝÀ» Á¦°øÇÑ´Ù. 5¡¯ RACE ¹æ¹ýÀº BCR heavy chain ¶Ç´Â light chainÀÇ constant regionÀ» ƯÀÌÀûÀ¸·Î Ÿ°ÙÇÒ ¼ö ÀÖ¾î, ÆíÇ⼺À» ÇöÀúÈ÷ ÁÙÀÏ ¼ö ÀÖ´Ù. Áï, º» Á¦Ç°À» ÀÌ¿ëÇϸé BCR Àü»çüÀÇ V(D)J °¡º¯¿µ¿ªÀ» ƯÀÌÀûÀ¸·Î ĸÃÄÇÏ¿© ÁõÆøÇÒ »Ó¸¸ ¾Æ´Ï¶ó, B-cell repertoire¸¦ ¸Å¿ì ¹Î°¨ÇÏ°í ÀçÇö¼º ³ô°Ô ºÐ¼®ÇÒ ¼ö ÀÖ´Ù.
º» Á¦Ç°Àº peripheral blood mononuclear cells (PBMCs)¿¡¼ ÃßÃâÇÑ 10 ng - 1 ¥ìg total RNA ¶Ç´Â B cell·ÎºÎÅÍ ÃßÃâÇÑ 1 - 100ng total RNA·ÎºÎÅÍ °íÇ°ÁúÀÇ Illumina
¢ç
-ready library¸¦ Á¦ÀÛ °¡´ÉÇϸç, Human immunoglobulin IgG, IgMÀÇ heavy chain°ú light chain (kappa and lamda)À» ¸ðµÎ ºÐ¼® °¡´ÉÇÏ´Ù. º» Á¦Ç°Àº PCR ¿À·ù, duplicate, ½ÃÄö½Ì ¿À·ù¿¡¼ ±âÀÎÇÏ´Â sequence reads¸¦ Á¦°ÅÇÒ ¼ö ÀÖ´Â unique molecular identifiers (UMI) ±â¼úÀ» Æ÷ÇÔÇÏ°í ÀÖ¾î º¸´Ù Á¤È®ÇÏ°í ½Å·Ú¼º ³ôÀº µ¥ÀÌÅ͸¦ ¾òÀ» ¼ö ÀÖ´Ù.
½ÃÄö½Ì ÀÌÈÄ¿¡´Â UMI-based error correctionÀ» À§ÇÑ
Takara Bio Immune Profiler Software
¸¦ ÀÌ¿ëÇÏ¿© µ¥ÀÌÅ͸¦ ºÐ¼®ÇÒ ¼ö ÀÖ´Ù. ÀÌ ¼ÒÇÁÆ®¿þ¾î´Â clonotype count, µ¿Á¤ ¹× V(D)J) ¿µ¿ªÀÇ ¼¿ ºÐ¼® µîÀ» Æ÷ÇÔÇÑ °íÇ°ÁúÀÇ BCR profiling ºÐ¼®À» Á¦°øÇÑ´Ù.
±×¸² 1. SMARTer Human BCR IgG IgM H/K/L Profiling Kit workflow
Panel A.
SMARTScribe Reverse Transcriptase´Â 1
st
strand cDNA°¡ ÇÕ¼ºÇϸç, mRNAÀÇ 5¡¯ ¸»´Ü¿¡ À̸£¸é nontemplated nucleotide (3¡¯-xxxxx-5¡¯)°¡ ÇÕ¼ºµÈ cDNAÀÇ 3¡¯ ¸»´Ü¿¡ ºÎ°¡µÈ´Ù. ÀÌ·¸°Ô ºÎ°¡µÈ nontemplated nucleotide¿¡ SMART UMI Oligonucleotide°¡ °áÇÕÇÏ°í, Ãß°¡ÀûÀÎ ¿ªÀü»ç ¹ÝÀÀ¿¡ ÀÇÇÏ¿© nontemplated nucleotide ¹× SMART UMI Oligonucleotide ¼¿°ú »óº¸ÀûÀÎ ¿°±â¸¦ cDNA¿¡ ºÎ°¡ ÇÕ¼ºÇÑ´Ù (Template-switching step). ÀÌ °úÁ¤¿¡¼ ÇÕ¼ºµÈ 1
st
strand cDNA´Â µÎ ´Ü°èÀÇ gene specific PCR amplification °úÁ¤À» °ÅÄ¡°Ô µÈ´Ù (Panel B ÂüÁ¶)
Panel B.
ù¹ø° ´Ü°èÀÇ PCR¿¡¼´Â 1
st
strand cDNA¸¦ ÁÖÇüÀ¸·Î ÇÏ¿©, Illumina Read Primer 2 ¼¿¿¡ »óº¸ÀûÀÎ ¿°±â¼¿À» °¡Áø forward primer (PCR1 Universal Forward Primer)¿Í BCR heavy/light chainÀÇ constant region¿¡ »óº¸ÀûÀÎ ¿°±â¼¿À» °¡Áø reverse primer (PCR1 IgG/IgM/IgK/IgL Reverse primers)À» ÀÌ¿ëÇÑ´Ù. Áï, ù¹ø° ´Ü°èÀÇ PCR¿¡¼´Â BCR Heavy chain ¶Ç´Â light chainÀÇ constant region ºÎÀ§¿Í °¡º¯¿µ¿ª Àüü¿¡ ´ëÇÑ ÁõÆø»ê¹°À» ¾òÀ» ¼ö ÀÖ´Ù. ¶ÇÇÑ µÎ¹ø° ´Ü°èÀÇ PCRÀº ù¹ø° PCR »ê¹°À» ÁÖÇüÀ¸·Î Çϸç, BCR heavy/light chainÀÇ constant region°ú °¡º¯¿µ¿ª Àüü ºÎºÐÀ» ÁõÆøÇϱâ À§ÇÑ semi-nested primers (PCR2 IgG/IgM/IgK/IgL Reverse primers¸¦ ÀÌ¿ëÇÑ´Ù.
±×¸² 2. Clonotype calls identified from 1-100 ng B-cell RNA
.
Human CD19+ B-cell total RNA 1 ng, 10 ng, 100 ngÀ¸·ÎºÎÅÍ BCR ProfilingÀ» À§ÇÑ IgG, IgM, IgK, IgL library¸¦ Á¦ÀÛÇÏ¿´´Ù. µ¥ÀÌÅÍ ºÐ¼®À» À§ÇØ RNA input 1 ng, 10 ng, 100 ng library¿¡ ´ëÇÏ¿© °¢°¢ 100,000 reads, 400,000 reads, 5,000,000 reads·Î downsampling ÇÏ¿´À¸¸ç, ±× ÀÌÈÄ Takara Bio Immmune Software¸¦ ÀÌ¿ëÇØ µ¥ÀÌÅÍ ºÐ¼®ÇÏ¿´´Ù.
±×¸² 3. Scatterplot showing reproducibility between libraries generated with 1 ¥ìg of PBMC RNA.
°Ç°ÇÑ °ø¿©ÀÚÀÇ PMBC·ÎºÎÅÍ ÃßÃâÇÑ 1 ¥ìg total RNA·ÎºÎÅÍ IgG, IgM, IgK, IgL¿¡ ´ëÇÑ library¸¦ duplicate·Î Á¦ÀÛÇÏ¿´´Ù. 13 million reads·Î downsampling ÇÏ°í clonotypeÀ» ºÐ¼®ÇÏ¿´´Ù. ±× °á°ú, ¸ðµç plot¿¡¼ 2°³ÀÇ library (IgG, IgM, IgK, IgL)¸¦ È®ÀÎÇÏ¿´À¸¸ç, ³ôÀº ÀçÇö¼ºÀ» º¸¿´´Ù (Pearson 0.98, Spearman 0.72)
±×¸² 4. Clonotype counts from PBMC RNA obtained from different donors.
SMARTer Human BCR IgG IgM H/K/L Profiling Kit¸¦ ÀÌ¿ëÇÏ¿© 8¸íÀÇ 10 ng PMBC RNA (°¢°¢ ´Ù¸¥ »ö»óÀ¸·Î Ç¥±â)·ÎºÎÅÍ IgG, IgM, IgK, IgL clonotypeÀ» ºÐ¼®ÇÏ¿´´Ù. ±× °á°ú, °¢°¢ÀÇ »ùÇø¶´Ù clonotype count°¡ ´Ù¸¥ ´Ù¾çÇÑ clonotypeÀ» °ËÃâÇÒ ¼ö ÀÖ¾ú´Ù.
±×¸² 5. Evaluation of sequencing saturation for 10-ng PBMC RNA libraries.
SMARTer Human BCR IgG IgM H/K/L Profiling Kit¸¦ ÀÌ¿ëÇÏ¿© ÇÑ ¸íÀÇ °ø¿©ÀÚÀÇ 10 ng PMBC RNA·ÎºÎÅÍ BCR profiling library¸¦ Á¦ÀÛÇÏ¿´´Ù.
Panel A.
UMI based-correction À¯¹«¿¡ µû¶ó °¢±â ´Ù¸¥ sequence depth Á¶°Ç¿¡¼ÀÇ IgG clonotype count¸¦ ³ªÅ¸³Â´Ù (Blue line : UMI-based correction ÁøÇà / Purple line : UMI-based correction ÁøÇàÇÏÁö ¾ÊÀ½). IgG¿Í ´Ù¸¥ »ç½½ (IgM, IgK, IgL - º» Ç¥¿¡ Ç¥ÇöµÇÁö ¾ÊÀ½)¿¡ ´ëÇÑ sequencing library´Â library´ç 1.5 million reads·Î ºÐ¼®ÇÏ¿´À¸¸ç, 100¸¸, 500K, 200K, 100K, 50K, 50K reads·Î downsamplingÇÏ¿´´Ù. ´Ù¾çÇÑ sequence depth¿¡ ´ëÇÑ ¼¿Àº Takara Bio Immune Profiler Software·Î ºÐ¼®ÇÏ¿´´Ù. UMI-based correctionÀ» ÀÌ¿ëÇÏ¿© º¸´Ù Á¤È®¼º ³ôÀº clonotype °ËÃâÀÌ °¡´ÉÇÏ´Ù.
Panel B.
°¢°¢ 100K, 1,500K sequencing depth¿¡ ´ëÇÑ overlapped clonotypeÀ» È®ÀÎÇÒ ¼ö ÀÖ´Ù.
Àû¿ë
Human BCR repertoire analysis (heavy and light [kappa and lambda] chains)
±¸¼ºÇ° (ÀÚ¼¼ÇÑ ³»¿ëÀº CoA¸¦ ÂüÁ¶Çϼ¼¿ä)
Package 1:
Control Total RNA(1 ¥ìg/§¡ )
SMART UMI Oligo
Package 2:
BCR Enhancer
First-Strand Buffer
dT Buffer
SMARTScribe Reverse Transcriptase(100 U/§¡)
RNase Inhibitor(40 U/§¡)
Elution Buffer(10 mM)
Nuclease-Free Water
PrimeSTAR GXL SP DNA Polymerase(1.25 U/§¡)
5X PrimeSTAR GXL Buffer
dNTP Mixture(2.5 mM each)
hBCR PCR1 Universal Forward
hBCR PCR1 IgG Reverse
hBCR PCR1 IgM Reverse
hBCR PCR1 IgK Reverse
hBCR PCR1 IgL Reverse
Package 3:
SMARTer
¢ç
Human BCR IgG IgM H/K/L Profiling Kit (Code 634466, 12ȸºÐ)
hBCR PCR2 IgG Reverse 1
hBCR PCR2 IgM Reverse 1
hBCR PCR2 IgK Reverse 1
hBCR PCR2 IgL Reverse 1
hBCR PCR2 Forward 1-12
SMARTer
¢ç
Human BCR IgG IgM H/K/L Profiling Kit (Code 634467, 48ȸºÐ)
hBCR PCR2 IgG Reverse 1-4
hBCR PCR2 IgM Reverse 1-4
hBCR PCR2 IgK Reverse 1-4
hBCR PCR2 IgL Reverse 1-4
hBCR PCR2 Forward 1-12
º¸Á¸
Package 1: - 70¡É
Package 2, package 3: -20 ¡É
BCR Enhancer, Nuclease-Free Water: -20¡É º¸Á¸. À¶ÇØ ÈÄ 4¡É º¸Á¸
Elution Buffer: -20¡É º¸Á¸. À¶ÇØ ÈÄ ½Ç¿Â º¸°ü .
±âŸ: - 20¡É
Keyword : BCR,B cell receptor,repertoire,variable region,Immune cell,Immune-seq,Immunology,UMI,Unique molecular index,Cogent.,SMART,smart-seq,NGS,Next generation sequencing,cancer,Cancer research,¾Ï¿¬±¸,vaccine,¹é½Å,¹é½Å¿¬±¸
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