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OMTO, Volume 24

Supplemental information

Isolation of TCR genes with tumor-killing activity

from tumor-infiltrating and circulating lymphocytes

in a tumor rejection cynomolgus macaque model

Koji Terada, Kenta Kondo, Hirohito Ishigaki, Ayaka Nagashima, Hiroki Satooka, Seiji

Nagano, Kyoko Masuda, Teruhisa Kawamura, Takako Hirata, Kazumasa

Ogasawara, Yasushi Itoh, Hiroshi Kawamoto, and Yasutoshi Agata

Supplemental Table S1. V(D)J genes and CDR3 amino acid sequences of the TCR genes isolated from CD8+PD-1+ T cells in TILs of Macaque

ID

TRBV

TRBD

TRBJ

CDR3 aa seq.

TRAV

TRAJ

CDR3 aa seq.

TCR pair

1.

TRBV7-2*01

TRBD2*01

TRBJ1-2*01

ASRPGGYDYT

TRAV23

TRAJ54*01

AAVSAQGAQKLV

5A9

2.

TRBV7-2*01

TRBD2*01

TRBJ1-2*01

ASRPGGYDYT

TRAV23

TRAJ54*01

AAVSAQGAQKLV

3.

TRBV7-2*01

TRBD2*01

TRBJ1-2*01

ASRPGGYDYT

TRAV23

TRAJ54*01

AAVSAQGAQKLV

4.

TRBV7-2*01

TRBD2*01

TRBJ1-2*01

ASRPGGYDYT

TRAV23

TRAJ54*01

AAVSAQGAQKLV

5.

TRBV7-2*01

TRBD2*01

TRBJ1-2*01

ASRPGGYDYT

TRAV23

TRAJ54*01

AAVSAQGAQKLV

6.

TRBV21-1*01

TRBD1*01

TRBJ1-2*01

ASSKGPQGTYDYT

TRAV13-1*01

TRAJ10*01

AASIEGGGNKLT

7.

TRBV21-1*01

TRBD1*01

TRBJ1-2*01

ASSKGPQGTYDYT

TRAV13-1*01

TRAJ10*01

AASIEGGGNKLT

8.

TRBV21-1*01

TRBD1*01

TRBJ1-2*01

ASSKGPQGTYDYT

TRAV13-1*01

TRAJ10*01

AASIEGGGNKLT

9.

TRBV21-1*01

TRBD1*01

TRBJ1-2*01

ASSKGPQGTYDYT

TRAV13-1*01

TRAJ10*01

AASIEGGGNKLT

10.

TRBV21-1*01

TRBD1*01

TRBJ1-2*01

ASSKGPQGTYDYT

TRAV13-1*01

TRAJ10*01

AASIEGGGNKLT

11.

TRBV2*01

TRBJ1-1*01

ASSELRNTEAF

TRAV36

TRAJ29*01

AVLNSGNRALV

12.

TRBV2*01

TRBJ1-1*01

ASSELRNTEAF

TRAV36

TRAJ29*01

AVLNSGNRALV

13.

TRBV2*01

TRBJ1-1*01

ASSELRNTEAF

TRAV36

TRAJ29*01

AVLNSGNRALV

14.

TRBV2*01

TRBJ1-1*01

ASSELRNTEAF

TRAV36

TRAJ29*01

AVLNSGNRALV

15.

TRBV5-6*01

TRBD2*02

TRBJ2-3*01

ASSLVRGLSDPQY

TRAV27*01

TRAJ52*01

AGAGGAGYGKL

16.

TRBV5-6*01

TRBD2*02

TRBJ2-3*01

ASSLVRGLSDPQY

TRAV27*01

TRAJ52*01

AGAGGAGYGKL

17.

TRBV5-6*01

TRBD2*02

TRBJ2-3*01

ASSLVRGLSDPQY

TRAV27*01

TRAJ52*01

AGAGGAGYGKL

18.

TRBV5-6*01

TRBD2*02

TRBJ2-3*01

ASSLVRGLSDPQY

TRAV27*01

TRAJ52*01

AGAGGAGYGKL

19.

TRBV2*01

TRBD2*01

TRBJ2-5*01

ASSEAGTPLGETQY

TRAV8-4*01

TRAJ31*01

AVNDRDNNARVI

20.

TRBV2*01

TRBD2*01

TRBJ2-5*01

ASSEAGTPLGETQY

TRAV8-4*01

TRAJ31*01

AVNDRDNNARVI

21.

TRBV2*01

TRBD2*01

TRBJ2-5*01

ASSEAGTPLGETQY

TRAV8-4*01

TRAJ31*01

AVNDRDNNARVI

5B1

4C2

4D3

3E5

22.

TRBV5-6*01

TRBD1*01

TRBJ1-5*01

ASSLAYRETYQPQY

TRAV25*01

TRAJ20*01

AGEAYNNYKLS

23.

TRBV5-6*01

TRBD1*01

TRBJ1-5*01

ASSLAYRETYQPQY

TRAV25*01

TRAJ20*01

AGEAYNNYKLS

24.

TRBV5-6*01

TRBD1*01

TRBJ1-5*01

ASSLAYRETYQPQY

TRAV25*01

TRAJ20*01

AGEAYNNYKLS

25.

TRBV5-1*01

TRBD2*01

TRBJ2-1*01

ASSLGWGELNEQF

TRAV8-6*02

TRAJ33*01

AVSGDSNYQLI

26.

TRBV5-1*01

TRBD2*01

TRBJ2-1*01

ASSLGWGELNEQF

TRAV8-6*02

TRAJ33*01

AVSGDSNYQLI

27.

TRBV5-5*01

TRBD1*01

TRBJ1-1*01

ASSLGQLNTEAF

TRAV26-1*01

TRAJ43*01

IGRFGNDIR

28.

TRBV5-5*01

TRBD1*01

TRBJ1-1*01

ASSLGQLNTEAF

TRAV26-1*01

TRAJ43*01

IGRFGNDIR

29.

TRBV10-1*01

TRBJ1-1*01

ASFSSLNTEAF

TRAV25*01

TRAJ22*01

SSIFCDSGWQLT

30.

TRBV10-1*01

TRBJ1-1*01

ASFSSLNTEAF

TRAV25*01

TRAJ22*01

SSIFCDSGWQLT

31.

TRBV10-1*01

TRBD2*01

TRBJ2-1*01

ASSEDWGGFYGEQF

TRAV38-2

TRAJ53*01

AYRGSSNYKLT

32.

TRBV10-1*01

TRBD2*01

TRBJ2-1*01

ASSEDWGGFYGEQF

TRAV38-2

TRAJ53*01

AYRGSSNYKLT

33.

TRBV12-3*01

TRBJ2-7*01

ASKKSSYEQY

TRAV6*01

TRAJ34*01

ALHNANKLI

34.

TRBV12-3*01

TRBJ2-7*01

ASKKSSYEQY

TRAV6*01

TRAJ34*01

ALHNANKLI

35.

TRBV2*01

TRBJ2-7*01

ASRLYEQY

TRAV5*01

TRAJ30*01

AENREDKII

36.

TRBV2*01

TRBD2*01

TRBJ2-4*01

PAVMRRGEAKTLST

TRAV19*01

TRAJ33*01

ALNEAEDSNYQLI

37.

TRBV2*01

TRBD1*01

TRBJ2-7*01

ATRTDLYEQY

TRAV20*01

TRAJ53*01

AVNSGSSNYKLT

38.

TRBV2*01

TRBD1*01

TRBJ2-3*01

ASSRTGGMNTDPQY

TRAV22*01

TRAJ32*01

AVLYYGGSGNKLI

39.

TRBV2*01

TRBJ1-5*01

ASSDLSNQPQY

TRAV26-1*01

TRAJ5*01

IVRPPDTGRRALT

40.

TRBV2*01

TRBJ1-1*01

ASSELRNTEAF

TRAV36

TRAJ29*01

CAQFRNRAFV

41.

TRBV3-1*01

TRBD2*01

TRBJ2-1*01

ASSQDGGADNEQF

TRAV9-1*01

TRAJ28*01

ALNLGYSGAGSYQLT

42.

TRBV3-1*01

TRBD2*02

TRBJ1-2*01

ASSQGRGNYDYT

TRAV18*01

TRAJ10*01

VLKGASNKLT

43.

TRBV3-2*01

TRBJ2-5*01

ASTSFGDAVETQY

TRAV6*01

TRAJ40*01

ALDASTTGNYKYI

3F1

1D4

44.

TRBV3-2*01

TRBD1*01

TRBJ1-4*01

ASSQQQATNEKLF

TRAV9-2*01

TRAJ6*01

ALTHPSGGGYVLT

45.

TRBV5-1*01

TRBJ1-4*01

ASSVNEKLF

TRAV12-3*01

TRAJ2*01

AMTLGGTIDKLT

46.

TRBV5-1*01

TRBD1*01

TRBJ2-3*01

ASSIRDRGEDPQY

TRAV23

TRAJ37*02

AASEASSNTGKLI

47.

TRBV5-5*01

TRBD1*01

TRBJ1-1*01

ASSLGQLNTEAF

TRAV26-1*01

TRAJ49*01

IVGFGNQL

48.

TRBV5-6*01

TRBD2*02

TRBJ2-1*01

ASSRLGGAHGEQF

TRAV4*01

TRAJ36*01

LVGDGSWGKQPV

49.

TRBV5-6*01

TRBD1*01

TRBJ1-5*01

ASSLEQGRNQPQY

TRAV12-3*01

TRAJ24*02

AMRADSWGKLQ

50.

TRBV5-6*01

TRBD1*01

TRBJ1-5*01

ASRQANREIYQPQY

TRAV25*01

TRAJ20*01

AGEASNNYKLS

51.

TRBV5-6*01

TRBD1*01

TRBJ2-3*01

ASTLAGRGRSHPQY

TRAV25*01

TRAJ20*01

AGEASNNYKLS

52.

TRBV5-6*01

TRBD1*01

TRBJ2-7*01

ASSRGRQTYEQY

TRAV38-1*01

TRAJ43*01

AFMIYNNNDIR

53.

TRBV5-8*01

TRBJ2-3*01

ASSLGHPPPQS

TRAV26-1*01

TRAJ43*01

IGRFGNDIR

54.

TRBV6-1*01

TRBD1*01

TRBJ2-5*01

ASSETGDQETQ

TRAV12-2*02

TRAJ22*01

AVYPDSGWQLT

55.

TRBV6-1*01

TRBD1*01

TRBJ1-6*02

ASSEYRVSYNSPLH

TRAV14

TRAJ30*01

AMMNRDDKII

56.

TRBV6-1*01

TRBD1*01

TRBJ2-4*01

ASTPGQTSQNTQY

TRAV20*01

TRAJ9*01

AVQAWTGGFKTV

57.

TRBV6-1*01

TRBD2*01

TRBJ1-3*01

KWPEVGGFRKHHV

TRAV21*01

TRAJ9*01

AVGYTGGFKTV

58.

TRBV6-1*01

TRBD1*01

TRBJ2-7*01

ASTKQGPQDEQY

TRAV29

TRAJ52*01

ASCGAGYGKLT

59.

TRBV6-5*01

TRBD1*01

TRBJ1-1*01

GSSEGVKTEAF

TRAV3*01

TRAJ40*01

AVNEVSGNYKYI

60.

TRBV6-5*01

TRBD2*01

TRBJ2-5*01

LSPSVTKRPST

TRAV3*01

TRAJ40*01

AVNEVSGNYKYI

61.

TRBV6-5*01

TRBJ2-6*01

GSSVSSGASVLT

TRAV18*01

TRAJ8*01

VVHRLSETR

62.

TRBV7-2*01

TRBJ2-1*01

ASSLLALNEQF

TRAV17*01

TRAJ48*01

VTEGFGNEKLT

63.

TRBV7-2*01

TRBD2*01

TRBJ1-2*01

ASRPGGYDYT

TRAV19*01

TRAJ52*01

ALNEESWWCWLWKAD

64.

TRBV7-2*01

TRBJ2-1*01

ASSLLALNEQF

TRAV26-2*01

TRAJ48*01

LLGRGVEVRNP

65.

TRBV7-2*01

TRBJ1-2*01

ASRDSMSMDDYT

TRAV29

TRAJ52*01

AARGSGYGKLT

66.

TRBV7-2*01

TRBD1*01

TRBJ2-5*01

ASSLVGSRVTETQY

TRAV38-1*01

TRAJ42*01

AFMKHEVYGGSQGNLI

67.

TRBV7-4*01

TRBD1*01

TRBJ2-3*01

ASSGRDRDLADPQY

TRAV18*01

TRAJ4*01

VLPAGGGYDKLI

68.

TRBV7-6*01

TRBD1*01

TRBJ2-7*01

ASSSLQTDWGDFYEQY

TRAV12-3*01

TRAJ37*02

AMSVTSNTGKLI

69.

TRBV7-6*01

TRBD1*01

TRBJ2-1*01

ASSPDWGEGSPSEQF

TRAV13-2*01

TRAJ47*02

AEMDYGNKLI

70.

TRBV7-6*01

TRBD2*01

TRBJ2-4*01

ASSPGPRRGAVNRLY

TRAV16*01

TRAJ54*01

ALSGSLGAQKLV

71.

TRBV7-9*01

TRBD2*02

TRBJ2-7*01

ASSGSQPSYEQY

TRAV13-1*01

TRAJ33*01

AATPGSNYQLI

1B9

1G9

72.

TRBV7-9*01

TRBD1*01

TRBJ2-4*01

ASSLGTPGNTQY

TRAV16*01

TRAJ16*01

ALKSSDGQKLL

73.

TRBV9*01

TRBJ2-2*01

PAANFMETPRSCS

TRAV26-1*01

TRAJ22*01

IVRPPSDSGWQLT

74.

TRBV9*01

TRBJ2-1*01

ASSEVGYGEQF

TRAV35*01

TRAJ32*01

AGQKGGGGSGNKLI

75.

TRBV9*01

TRBD1*01

TRBJ2-1*01

ATGELGNGDQF

TRAV35*01

TRAJ32*01

AGQKGGGGSGNKLI

76.

TRBV10-1*01

TRBJ2-1*01

ASSEINALYGEQF

TRAV8-6*02

TRAJ24*02

AVTTDSWGKLQ

77.

TRBV10-1*01

TRBJ1-1*01

ASFSSLNTEAF

TRAV25*01

TRAJ22*01

SSISSDSGWQLT

78.

TRBV10-1*01

TRBJ1-1*01

ASFSSLNTEAF

TRAV25*01

TRAJ22*01

STIYCGCGSQLT

79.

TRBV12-3*01

TRBD1*01

TRBJ2-7*01

ASTTPGQVSEQY

TRAV22*01

TRAJ40*01

AVTTGNYKYI

80.

TRBV12-3*01

TRBD1*01

TRBJ2-1*01

ASSLKWGSSYNEQF

TRAV23

TRAJ27*01

AAGDNAGKLT

81.

TRBV13*01

TRBJ2-7*01

ASNFVSIYEQY

TRAV2*01

TRAJ27*01

AVLHTNADKLT

82.

TRBV13*01

TRBD1*01

TRBJ1-1*01

TSREGRGLGNEAF

TRAV9-1*01

TRAJ27*01

ALEAGKLT

83.

TRBV14*01

TRBD2*02

TRBJ1-3*01

ASSQAEGTISGNTVY

TRAV2*01

TRAJ11*01

AVEDSGYSTLT

84.

TRBV14*01

TRBD2*02

TRBJ2-3*01

ASSLWEGRDPQY

TRAV2*01

TRAJ23*01

AVEDQDQAGKLI

85.

TRBV14*01

TRBD1*01

TRBJ1-5*01

ASSQGGGSQPQY

TRAV3*01

TRAJ34*01

AVRDPNANKLI

86.

TRBV14*01

TRBD1*01

TRBJ1-2*01

ASTSRDIHDYT

TRAV17*01

TRAJ44*01

ATDSFTGIASKLT

87.

TRBV14*01

TRBD1*01

TRBJ1-5*01

PGGRVAISPS

TRAV19*01

TRAJ32*01

APAGGYGGRGNKLI

88.

TRBV15*01

TRBD1*01

TRBJ2-4*01

ASSKDMGTEGTKTFSS

TRAV26-1*01

TRAJ48*01

IVRARQGGNENLT

89.

TRBV15*02

TRBD1*01

TRBJ1-2*01

ASSKKGQVSHST

TRAV16*01

TRAJ54*01

AQRVITQGAQKLV

90.

TRBV20-1*01

TRBD2*01

TRBJ2-7*01

SAPGLASSYEQY

TRAV9-1*01

TRAJ31*01

ALRQNNNARVI

91.

TRBV20-1*01

TRBD1*01

TRBJ1-2*01

SATLGTEFFRDYT

TRAV12-1*01

TRAJ23*01

AVNMAYNQAGKLI

92.

TRBV20-1*01

TRBD1*01

TRBJ1-3*01

SARMQGTRITVY

TRAV38-2

TRAJ33*01

AYRGNMDSNYQLI

93.

TRBV23-1*01

TRBD1*01

TRBJ2-3*01

ASTGFGDPQY

TRAV9-2*01

TRAJ13*01

ALSSGSYQKVT

94.

TRBV23-1*01

TRBD1*01

TRBJ2-3*01

ASTGFGDPQY

TRAV9-2*01

TRAV9-2*01

ALSSGSTRESS

95.

TRBV23-1*01

TRBD1*01

TRBJ2-3*01

ASKQGWGHRSSV

TRAV18*01

TRAJ33*01

VLRDPMDSNYQLI

96.

TRBV23-1*01

TRBJ2-3*01

ASSQGWGTDPQY

TRAV18*01

TRAJ33*01

VLRDPMDSNYQLI

97.

TRBV24-1*01

TRBD1*01

TRBJ1-2*01

ATSDLSITGADYDYT

TRAV12-3*01

TRAJ32*01

ARPGGSGNKLI

98.

TRBV27*01

TRBD2*01

TRBJ2-1*01

ASSGGPGQF

TRAV1-2*01

TRAJ34*01

AVRANKL

99.

TRBV27*01

TRBD1*01

TRBJ1-1*01

ASSKGGGNTEAF

TRAV6*01

TRAJ24*02

APGSDSWGKLQ

100.

TRBV27*01

TRBD1*01

TRBJ1-5*01

ASTPLGESNQPQY

TRAV8-4*01

TRAJ11*01

AVILNSGYSTLT

101.

TRBV27*01

TRBJ2-1*01

ASSLRDFDGEQF

TRAV25*01

TRAJ21*01

AGMDNFNKFY

102.

TRBV27*01

TRBJ1-3*01

ASSSWQPSYSGNTVY

TRAV29

TRAJ30*01

GGRDDKII

103.

TRBV28*01

TRBD1*01

TRBJ1-1*01

ASSFWRGVNTEAF

TRAV8-3*02

TRAJ12*01

HNGATGDNKLI

104.

TRBV28*01

TRBD1*01

TRBJ1-1*01

ASSFWRGVNTEAF

TRAV8-3*01

TRAJ50*01

GAGATSYNKLT

105.

TRBV28*01

TRBJ2-6*01

ASLDSGASVLT

TRAV12-3*01

TRAJ41*01

AMTHSNSGYALN

106.

TRBV29-1*01

TRBD1*01

TRBJ1-2*01

SVGTGYNYDYT

TRAV4*01

TRAJ30*01

LVGERDDKII

107.

TRBV29-1*01

TRBJ2-1*01

SVASLGFSYGEQF

TRAV12-3*01

TRAJ43*01

AMKKDNNDIR

108.

TRBV30*02

TRBD1*01

TRBJ1-1*01

AWKYEGGQTEAF

TRAV36

TRAJ29*01

AYLSGNRALV

Supplemental Table S2. Nucleotide sequence of PCR primers and PCR conditions.

Primer name

TCRb primer sequence

5A9_b_F

5’-TTCAGTCGACTGGATCCAGCCGCCATGGGCACCAGGCTCCT-3’

5A9_b_R

5’-GGTCACGTTCCTCAGGTCAACAGTTAACTTGGTCCCTG-3’

5B1_b_F

5’-TTCAGTCGACTGGATCCAGCCACCATGTGCCTCAGACTTCT-3’

5B1_b_R

5’-GGTCACGTTCCTCAGGTCCTCTACAACAGTTAACTTG-3’

4C2_b_F

5’-TTCAGTCGACTGGATCCAGCCGCCATGGATACCTGTCTCCT-3’

4C2_b_R

5’-GGTCACGTTCCTCAGGTCCTCTACAACTGTGAGTCTGG-3’

4D3_b_F

5’-TTCAGTCGACTGGATCCAGCCGCCATGGGCCCCGGGCTCCT-3’

4D3_b_R

5’-GGTCACGTTCCTCAGGTCCTCGAGCACTGTCAGCCGGG-3’

3E5_b_F

5’-TTCAGTCGACTGGATCCAGCCGCCATGGATACCTGGCTCCT-3’

3E5_b_R

5’-GGTCACGTTCCTCAGGTCCTCGAGCACCAGGAGCCGCG-3’

3F1_b_F

5’-TTCAGTCGACTGGATCCAGCCGCCATGGGCCCCGGGCTCCT-3’

3F1_b_R

5’-GGTCACGTTCCTCAGGTCCTCTAGGACGGAGAGTCGAG-3’

1B9_b_F

5’-TTCAGTCGACTGGATCCAGCCGCCATGCTGTTTTGCTGGGCA-3’

1B9_b_R

5’-GGTCACGTTCCTCAGGTCCTCGAGCACTGTCAGCCGGG-3’

1D4_b_F

5’-TTCAGTCGACTGGATCCAGCCACCATGGATACCTGGCTCCT-3’

1D4_b_R

5’-GGTCACGTTCCTCAGGTCCTCTAGGACGGAGAGTCGAG-3’

1G9_b_F

5’-TTCAGTCGACTGGATCCAGTTCCCCTTTCATCAATGC-3’

1G9_b_R

5’-GGTCACGTTCCTCAGGTCCTCTAGCACCAGGAGCCGCG-3’

1385_b_F

5’-TTCAGTCGACTGGATCCAGCCGCCATGGGCACCAGGCTCCTCTG-3’

1385_b_R

5’-GGTCACGTTCCTCAGGTCCTCTACGACCGTGAGCCTGG-3’

NYESO1_1G4_b_F

5’-TTCAGTCGACTGGATCCAGCCACCATGTCTATCGGCCTGCTG-3’

NYESO1_1G4_b_R

5’-GGTCACGTTCCTCAGGTCTTCCAGCACGGTCAGTCTGC-3’

TCRa primer sequence

5A9_a_F

5’-GAGGAGAATCCTGGCCCAATGGACAAGATCTTAGGAGC-3’

5A9_a_R

5’-CGGGCTCTGGGTTCTGGATATTCAGGTTGATGCTCAGCC-3’

5B1_a_F

5’-GAGGAGAATCCTGGCCCAATGACATCCATTCGAGCTGT-3’

5B1_a_R

5’-CGGGCTCTGGGTTCTGGATATTCAGTTCCACTTTTAGCT-3’

4C2_a_F

5’-GAGGAGAATCCTGGCCCAATGATGAAGTGTTCACAGGC-3’

4C2_a_R

5’-CGGGCTCTGGGTTCTGGATATTTGGAATCACAGAAAGTC-3’

4D3_a_F

5’-GAGGAGAATCCTGGCCCAATGGTCCTGAAATTCTCCGT-3’

4D3_a_R

5’-CGGGCTCTGGGTTCTGGATATTTGGATAGACAGTTAAGA-3’

3E5_a_F

5’-GAGGAGAATCCTGGCCCAATGCTCCTGCTGCTCGTCCT-3’

3E5_a_R

5’-CGGGCTCTGGGTTCTGGATATTGGGCTTCACCACCAGCT-3’

3F1_a_F

5’-GAGGAGAATCCTGGCCCAATGCACACATCTGCTTTCCA-3’

3F1_a_R

5’-CGGGCTCTGGGTTCTGGATATTTGCTCTTACAGTTACTG-3’

1B9_a_F

5’-GAGGAGAATCCTGGCCCAATGGACAAGATCTTAGGAGC-3’

1B9_a_R

5’-CGGGCTCTGGGTTCTGGATATCTGGTTGTACTTGTAAAG-3’

1D4_a_F

5’-GAGGAGAATCCTGGCCCAATGAGGCTGGTGGCAAGAGT-3’

1D4_a_R

5’-CGGGCTCTGGGTTCTGGATATTTGGTTGCACTTGGAGTC-3’

1G9_a_F

5’-GAGGAGAATCCTGGCCCAATGATGAAATCCTTGAGAGT-3’

1G9_a_R

5’-CGGGCTCTGGGTTCTGGATATCAGGTACAACGGTCAATT-3’

1385_a_F

5’-GAGGAGAATCCTGGCCCAATGAAGACACTTACTGGATC-3’

1385_a_R

5’-CGGGCTCTGGGTTCTGGATATTTGGTTGCACTTGGAGTC-3’

NYESO1_1G4_a_F

5’-GAGGAGAATCCTGGCCCAATGGAAACCCTGCTGGGCCT-3’

NYESO1_1G4_a_R

5’-CGGGCTCTGGGTTCTGGATGTAGGGGTGCACGATCAGGC-3’

RT primer sequence

RTa

5’-CAGCGGTGTTTGGCAGCTCTTC-3’

RTb1

5’-CTGGCAAAAGAAGAATGTAT-3’

RTb2

5’-ACACAGATTGGGAGCAGGTA-3’

First PCR primer sequence

F1st

5’-TCAAGCAGTAGCAGCAGTTCGATAAGGATCCCCCCCCCCCCCDN-3’

R1sta

5’-GAGAGGGAGAGGAGGGGCGAT-3’

R1stb1

5’-TCACAACGGGTTAGAAGCTC-3’

R1stb2

5’-GGATGAAGAATGACCCGGGAT-3’

Second PCR primer sequence

F2nd

5’-AGCAGTAGCAGCAGTTCGATA-3’

R2nda

5’-AGTAAATAGGCAGACAGAGGT-3’

R2ndb1

5’-GTGGTCGGGGTAGAAGCCTGT-3’

R2ndb2

5’-GTGGTCGGGGTAGAAGCCTGT-3’

First PCR condition

1) 94˚C, 2 min

2) 98˚C, 10 sec

3) 68˚C, 30 sec (-2˚C / cycle)

4) 68˚C, 1 min 30 sec

5) Go to 2), Repeat 4 times

6) 98˚C, 10 sec

7) 60˚C, 30 sec

8) 68˚C, 1 min 30 sec

9) Go to 6), Repeat 24 times

10) 68˚C 10 min

11) 12˚C

Second PCR condition

1) 94˚C, 2 min

2) 98˚C, 10 sec

3) 68˚C, 30 sec (-2˚C / cycle)

4) 68˚C, 1 min 30 sec

5) Go to 2), Repeat 4 times

6) 98˚C, 10 sec

7) 60˚C, 30 sec

8) 68˚C, 1 min 30 sec

9) Go to 6), Repeat 29 times

10) 68˚C 10 min

11) 12˚C

...