- TCR repertoire profiling : 5' RACE¹ýÀ» Á¢¸ñ½ÃÄÑ TCR-α , TCR-β chainÀÇ full-length V(D)J variable region ¼¿À» ºÐ¼®ÇÒ ¼ö ÀÖ´Ù. V(D)J ¼¿ ºÐ¼® ½Ã Miseq ÃßõÇϸç, CDR3 ¼¿ ºÐ¼® ½Ã ¸ðµç Illumina® ±â±â¿¡ Àû¿ëÀÌ °¡´ÉÇÏ´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. (¿¹: NovaSeq™ Àû¿ë °¡´É)
- ´Ù¾çÇÑ input range : PBMCsÀÇ high-integrity RNA (10 ng-1 §¶), T cells total RNA (1-100 ng), whole T cell (1,000 - 10,000 purified cells), whole blood total RNA (20-200 ng)
- ³ôÀº Á¤È®µµ : UMIs ±â¹ÝÀ¸·Î PCR duplicates, errors º¸Á¤
- End-to-end solution : Cogent NGS Immune Profiler Software ·Î sequencing data¸¦ ºÐ¼®Çϰí Cogent NGS Immune Viewer ·Î °á°ú data¸¦ visualization
Á¦Ç°¼³¸í
SMART-Seq® Human TCR (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°¨Çϰí Á¤È®ÇÑ TCR 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 human TRA¿Í TRB À¯ÀüÀÚÀÇ V(D)J variable regionÀ» ¿ÏÀüÇÏ°Ô Ä¸Ã³ÇÑ´Ù. TCR-α, TCR-β chainÀ» µ¿½Ã¿¡ ¶Ç´Â °¢°¢ÀÇ chain¿¡ ´ëÇÑ NGS library¸¦ Á¦ÀÛÇÏ¿© ºÐ¼®ÇÒ ¼ö ÀÖ´Ù.
º» Á¦Ç°À» ÀÌ¿ëÇϸé RNA »ùÇà (RIN°ª 8 ÀÌ»ó)·ÎºÎÅÍ °íǰÁúÀÇ library¸¦ Á¦ÀÛÇÒ ¼ö ÀÖÀ¸¸ç, peripheral blood leukocytes (10 ng - 1 §¶), T cell À¯·¡ total RNA (1 - 100 ng), whole T cell (1,000 - 10,000 purified cells)¿¡ Àû¿ëÇÒ ¼ö ÀÖ´Ù.
»ùÇÃ
|
»ùÇ÷®
|
PBMC À¯·¡ total RNA
|
10 ng - 1 §¶
|
T-cell À¯·¡ total RNA
|
1 - 100 ng
|
ÀüÇ÷ À¯·¡ total RNA
|
20 - 200 ng
|
T-cell
|
1,000 - 10,000 cells
|
Á¦Ç° ³»¿¡´Â Unique Molecular Identifiers (UMIs)¸¦ Æ÷ÇÔÇϰí 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 ÆÇ¸Å)¸¦ »ç¿ëÇϱ⿡ index hopingÀ¸·Î ÀÎÇÑ ´Ù¸¥ library »ùÇà À¯·¡ÀÇreads ¿À¿°À» ¹æÁöÇÒ ¼ö ÀÖ´Ù.
Sequencing ÈÄ, UMI-based error correctionÀ» À§ÇØ Cogent NGS Immune Profiler Software ¶Ç´Â Cogent NGS Immune Viewer ¸¦ ÀÌ¿ëÇÏ¿© µ¥ÀÌÅ͸¦ ºÐ¼®ÇÒ ¼ö ÀÖ´Ù. º» ¼ÒÇÁÆ®¿þ¾î´Â clonotype count, µ¿Á¤ ¹× V(D)J ¿µ¿ªÀÇ ¼¿ ºÐ¼® µîÀ» Æ÷ÇÔÇÑ °íǰÁúÀÇ TCR profiling ºÐ¼®ÅøÀ» Á¦°øÇÑ´Ù.

±×¸² 1. SMART-Seq® Human TCR (with UMIs)À» ÀÌ¿ëÇÑ library prep 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°¡ °áÇÕÇϰí, 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°¡ TCR Àü»çü¿¡ mappingµÈ´Ù.

±×¸² 2. ´Ù¾çÇÑ RNA input ¾ç¿¡¼ ³ôÀº °¨µµ ¹× ÀçÇö¼ºÀ¸·Î clonotype °ËÃâ
TRA, TRB library´Â 1, 10, 100 ngÀÇ human CD3+ T cell total RNA¿Í 1,000 CD3+ T cells, 10,000 CD3+ T cells·ÎºÎÅÍ Á¦ÀÛÇÏ¿´´Ù. ¾ò¾îÁø sequence reads´Â Cogent NGS Immune Profiler Software·Î ºÐ¼®ÇÏ¿´´Ù.
% Jurkat RNA spiked in to 100 ng of PBMC RNA
|
Total read count (TRA/TRB)
|
Without UMI collapse
|
With UMI collapse
|
# of TRB raw reads
|
# of reads for TRBV12-3-TRBJ1-2
|
Detected percentage of Jurkat reads
|
# of detected UMIs
|
# of UMIs for TRBV12-3-TRBJ1-2
|
Detected percentage of Jurkat UMIs
|
10.0%
|
2,500,000
|
1,565,005
|
397,179
|
25.0%
|
281,280
|
62,629
|
22.0%
|
1.0%
|
2,500,000
|
1,422,102
|
47,160
|
3.3%
|
219,776
|
6,426
|
2.9%
|
0.1%
|
2,500,000
|
1,366,127
|
5,412
|
0.4%
|
189,580
|
631
|
0.33%
|
0.01%
|
2,500,000
|
1,218,025
|
521
|
0.043%
|
196,615
|
74
|
0.038%
|
0.001%
|
2,500,000
|
1,331,465
|
909
|
0.068%
|
197,870
|
6
|
0.003%
|
0.0001%
|
2,500,000
|
1,409,199
|
-
|
0%
|
124,149
|
-
|
0%
|
0%
|
2,500,000
|
1,222,245
|
-
|
0%
|
197,933
|
-
|
0%
|
Ç¥ 1. SMARTer® Human TCR a/b Profiling Kit v2 (TCRv2)ÀÇ °¨µµ¿Í ÀçÇö¼º Æò°¡
Leukemic Jurkat T cells (containing TRBV12-3-TRBJ1-2 clonotypes)ÀÇ homogeneous population À¸·ÎºÎÅÍ ¾òÀº RNA¸¦ ´Ù¾çÇÑ ³óµµ (10%, 1%, 0.1%, 0.01%, 0.001%, 0.0001%)·Î spikeÇÑ PBMC RNA»ùÇÿ¡ ´ëÇØ ¹Ýº¹ÀûÀÎ ºÐ¼®À» ÇÏ¿´´Ù. 100 ng total RNA·ÎºÎÅÍ TRB CDR3 ¿µ¿ªÀ» ÁõÆøÇϰí, Illumina NextSeq® ½Ã½ºÅÛÀ¸·Î 2 x 150 bpÀÇ reads¸¦ ¾ò¾ú´Ù. Sequencing reads´Â 2.5M reads·Î downsamplingµÇ¾ú´Ù. UMI¿¡ ÀÇÇÑ data¸¦ º¸Á¤ÇÏÁö ¾ÊÀº °æ¿ì (Without UMI collapse)¿Í UMI¿¡ ÀÇÇØ data º¸Á¤À» ÇßÀ» °æ¿ì (With UMI collapse) spike-in RNA ³óµµÀÇ °ËÃâ ÇѰ踦 À½¿µ (gray)À¸·Î ³ªÅ¸³»°í ÀÖ´Ù. UMI¿¡ ÀÇÇÑ data º¸Á¤ÀÌ ¾ø´Â °æ¿ì, TRBV12-3ÀÇ °ËÃâ ÇѰè´Â 0.01%¿´Áö¸¸, UMI¿¡ ÀÇÇØ data¸¦ º¸Á¤ÇÒ °æ¿ì¿¡´Â 0.001%±îÁö °ËÃâÇÒ ¼ö ÀÖ¾ú´Ù.

±×¸² 3. Ÿ»ç Á¦Ç°°úÀÇ ºñ±³
µÎ Á¾·ù °ËüÀÇ PBMC¿¡¼ total RNA¿Í gDNA¸¦ Á¤Á¦ÇÑ ÈÄ, 1.6 §¶ gDNA (Y»ç)¿Í 100 ng RNA (X»ç)ÀÇ library¸¦ Á¦ÀÛÇÏ¿´´Ù. °¢ Á¦Ç°À» »ç¿ëÇÏ¿© ¾òÀº TCR α/β libraryÀÇ clonotype ¼ö¸¦ ºñ±³ÇÑ °á°ú, ´ç»ç kit´Â TRA¸¦ Æò±Õ 48.7K, TRB¸¦ 163K ¾òÀ» ¼ö ÀÖ¾ú°í, Ÿ»ç´ëºñ ´õ ¸¹Àº clonetypeÀ» °ËÃâÇÒ ¼ö ÀÖ¾ú´Ù (NT: not tested).
±¸¼ºÇ° (ÀÚ¼¼ÇÑ ³»¿ëÀº CoA¸¦ ÂüÁ¶Çϼ¼¿ä)
Box 1:
- Control RNA ¡Ø (1 §¶/§¡)
- TCR SMART UMI Oligo (48 μM)
Box 2:
- 10X Lysis Buffer
- 5X Ultra Low First-Strand Buffer
- TCR dT Primer (12 μM)
- SMARTScribe Reverse Transcriptase (100 U/§¡)
- RNase Inhibitor (40 U/§¡)
- Elution Buffer (10 mM)
- Nuclease-Free Water
- PrimeSTAR GXL DNA Polymerase (1.25 U/§¡)
- 5X PrimeSTAR GXL Buffer
- dNTP Mix (2.5 mM each)
- hTCR PCR1 Universal Forward (12 μM)
- hTCRa PCR1 Reverse (12 μM)
- hTCRb PCR1 Reverse (12 μM)
- hTCRa PCR2 UDI Reverse (12 μM)
- hTCRb PCR2 UDI Reverse (12 μM)
¡Ø Control RNA is human spleen Total RNA.
º¸Á¸
Box 1: -70¡É
±× ¿Ü: -20¡É (À¶ÇØ ÈÄ¿¡´ÂBox 2ÀÇ 10X Lysis Buffer´Â 4¡É º¸°ü, Elution Buffer´Â ½Ç¿Â º¸°ü)
°ü·Ã Á¦Ç°
- Multiplexed sequencing library Á¦ÀÛÀ» À§ÇÑ Unique Dual Index Kit (Code 634756 ¿Ü)
- Complete BCR repertoire profiling SMART-Seq® Human BCR (with UMIs) (Code 634777 ¿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)
Citation
‘SMARTer® Human TCR a/b Profiling Kit v2’ Á¦Ç°Àº ‘SMART-Seq® Human TCR (with UMIs)’À¸·Î Á¦Ç°¸íÀÌ º¯°æµÇ¾ú½À´Ï´Ù (ÀÚ¼¼ÇÑ ³»¿ëÀº Á¦Ç°ÆäÀÌÁö¸¦ ÂüÁ¶). CitationÀÌ µ¿ÀÏÇÕ´Ï´Ù.
Myelodysplastic Syndrome Progression to Acute Myeloid Leukemia at the Stem Cell Level
Nature medicine | 2018 Oct 31 | PubMed ID: 30510255 | Read Article
An immunodominant NP 105-113 -B*07:02 cytotoxic T cell response controls viral replication and is associated with less severe COVID-19 disease
Nature immunology | 2021 Oct 27 | PubMed ID: 34853448 | Read Article
Polyclonal expansion of TCR Vbeta 21.3 + CD4 + and CD8 + T cells is a hallmark of Multisystem Inflammatory Syndrome in Children
Science immunology | 2022 Feb 04 | PubMed ID: 34035116 | Read Article
Clinical outcome following checkpoint therapy in renal cell carcinoma is associated with a burst of activated CD8 T cells in blood
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Epstein–Barr Virus+ B Cells in Breast Cancer Immune Response: A Case Report
Frontiers in Immunology | 2021 Nov 16 | PubMed ID: 34868006 | Read Article
Functional changes in cytotoxic CD8+ T-cell cross-reactivity against the SARS-CoV-2 Omicron variant after mRNA vaccination
Frontiers in Immunology | 2023 Jan 04 | Read Article
Rapid identification of CMV-specific TCRs via reverse TCR cloning system based on bulk TCR repertoire data
Frontiers in Immunology | 2022 Nov 18 | Read Article
Features of repertoire diversity and gene expression in human cytotoxic T cells following allogeneic hematopoietic cell transplantation
Communications Biology | 2021 Oct 11 | PubMed ID: 34635773 | Read Article
Assay and Isolation of Single Proliferating CD4+ Lymphocytes Using an Automated Microraft Array Platform
IEEE Transactions on Biomedical Engineeri… | 2020 Aug 01 | Read Article
Abstracts from the 53rd European Society of Human Genetics (ESHG) Conference: Interactive e-Posters
European Journal of Human Genetics | 2020 Dec 01 | PubMed ID: 33262485 | Read Article
Staphylococcus aureus Isolated from Skin from Atopic-Dermatitis Patients Produces Staphylococcal Enterotoxin Y, Which Predominantly Induces T-Cell Receptor Vα-Specific Expansion of T Cells
Infection and Immunity | 2020 Feb 01 | PubMed ID: 31740530 | Read Article
Genomic profiling of intestinal T-cell receptor repertoires in inflammatory bowel disease.
Genes and immunity | 2021 Jan 06 | PubMed ID: 32029881 | Read Article
Clinical and immunological response to checkpoint therapy in renal cell carcinoma is associated with TCF1+ CD8 T-cells in the tumor
ppr0375699 | 2021 Jul 28 | Read Article
Intratumoral mregDC and CXCL13 T helper niches enable local differentiation of CD8 T cells following PD-1 blockade
bioRxiv | 2022 Jun 26 | Read Article
Superantigenic TCR Vbeta 21.3 signature in Multisystem Inflammatory Syndrome in Children
medRxiv | 2021 Feb 12 | Read Article
Human thymopoiesis selects unconventional CD8 + α/β T cells that respond to multiple viruses
bioRxiv | 2020 Jul 29 | Read Article
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