Supplementary Materials and Methods
PCR primers, PCR and DHPLC annealing temperatures
Size Temperature (°C)
Gene Primera Primer sequence (5’à3’) (bp) PCR DHPLC
ADAMTS1 F gggtagttttaggatagggaaatgt 285 54.0 57.4
R tataataaaccaataaaacccccac
AOF2 F aggttggttttgggatagtagg 599 58.0 57.0
R accattttccaatcaaaaaaataaa
ARID5A F gttttgtagggaggtttaaggtgt 358 56.0 55.1
R acaacaaattaactaaacccaactc
BLOC1S3 F ggttgggaatttaggatt 527 54.0 56.8
R ccaacctactaaccaaaa
BMP3 F gagggaaggtatagatagattttg 346 58.0 56.8
R ccccaaccataactaaataccc
RT-PCR-F tggattgaacctcggaattgc 172 56.0
RT-PCR-R tctggatggtagcatgatttgatg
BMP4 F ttttgaggtttagagagagtgagg 485 59.0 56.6
R gagatggggagagaatatttagttg
BNIP3 F tgatatttggatttggattaataatgtt 601 56.0 54.5
R ccatcctactaataaaaaaactaaaac
RT-PCR-F atgagtctggacggagta 105 54.3
RT-PCR-R tgctatgggtatctgtttc
BOP1 F ggtttttttgattaagttgtggattt 648 53.0 55.6
R cataaaaaataaaaaacaaaactcctctta
C9orf21 F atttgttaggttttatttttgta 422 54.3 58.5
R actaaacaaactaccttccc
CAMTA1 F gggataggtaattatgagttatt 666 54.3 55.0
R ctaaacaaaacttcctaataacc
CBX1 F gtttttggtttatatgttttaatattttagt 421 55.0 58.3
R aattttttattttaaaataat
CCDC88A F ttggggagaaggtatgttttataga 601 58.0 58.3
R ctaaaaatccctaacacaaaatcac
CDKN2A F tttttagaggatttgagggatagg 392 65.0à50.0 57.6
R ctacctaattccaattcccctacaaacttc
COL2A1 ML-F tctaacaattataaactccaaccaccaa 91 56.0
ML-R gggaagatgggatagaagggaatat
ML-Probe 6FAM-ccttcattctaacccaatacctatcccacctctaaa-TAMRA
COPS3 F ttaggttgtttttatggaaatagtaa 342 56.4 56.2
R tcaaaactaaaaaaactattccaac
COX2 F tttggagaggaagttaagtgttt 605 54.0 53.0
R gccaaatactcacctatataacta
CUL7 F tttttttagttttgattttgggaaa 423 53.0 56.5
R taaaactctaaccacctcaaaaatc
DAB2IP F gggtattgggggtaaatagagtaga 345 53.9 54.0
R gaaaggggtttgtttgtaggaatat
DNAJB6 F gggtttttttaaattgataag 260 48.0 57.4
R aaaaacccattctaaaaacc
EIF2B3 F attttttgaggaaagaggttgagtt 363 58.5 55.2
R acctaacaaaacaaaaatcacaa
EPHA1 F tttttaaggaggtgaattaggtgag 480 56.0 54.2
R ccactcccaaaaacaaattactaac
ECEL1 F ggttatgttggtttttaagtatttgg 628 57.0 57.9
R aaaaacccctaaacaaaaccactac
ELK1 F gtatagttttgtagggaagggtt 301 58.5 55.0
R acccaaaactcacctatatataa
ELK3 F atttttaggggttgaggtgagga 398 58.5 57.1
R ctcctctttttttttcccctctaat
ELK4 F tgtagtgttttgggatttggtat 459 58.9 57.6
R actactcctaaatccctcccaa
ETS1 F gggtgattaagtttttaagaatg 604 52.8 57.7
R cttcccaaaaatctcaaattc
ETV5 F aagtttgtgttgattggttaatggt 575 62.0 58.0
R aaaaaaatcaccccaactactacac
FHIT F gtttaagtggaatattgtttttggg 399 53.0 53.9
R accccttaaaaaaatccaaaactac
FOXF1 F tttaaggttaagaagattaa 468 57.7 56.5
R aaattcttccaaaccctaat
GAPDH RT-PCR-F gagatggtgatgggatttc 226 62.0
RT-PCR-R gaaggtgaaggtcggagt
GATA4 F ggagattttagagtttggat 385 52.8 57.1
R ctcccactaactacctctct
GFRA1 F ggtgttggaaattttttaaagg 522 55.0 55.5
R aaaacacttcttccttccacat
ML-F ggattagtttcgcgtcggta 158 58.5
ML-R cgccccaattttaatacgaa
ML-Probe 6FAM- aacgcgaaatacaaaatcgc-TAMRA
RT-PCR-F gatatattccgggtggtcccattc 150 58.5
RT-PCR-R ggtgcacggggtgatgtacgc
HOXA10 F agagagtttagttaggaggattgtt 471 53.0 54.0
R tcccctttttctattataaaaaaaa
HOXA13 F ggtagaattatttgatttgt 337 55.4 55.4
R ccttccatattcttattaaact
HOXD10 F ggatagtgaattgttggtaaagaat 529 59.0 55.1
R tctcaactctaaaaacctccaaat
ITGB8 F ggggttagagtaggattatt 416 52.8 55.0
R cattaattacaactttacaacc
JMJD1C F ttgtgattagagtggaattattat 453 56.0à48.0 55.7
R ctcctttaaactcccaaattc
KCNF1 F agaagagggtatagtttgaaaa 711 62.0à47.0 58.3
R caaccaaacaattaataaactcc
KCNH1 F tgaaagttgtttataatgtttttttt 511 56.0 56.8
R cttcccaaaaatctcaaattc
RT-PCR-F cgaagatggtggcatagagaa 203 52.4
RT-PCR-R cccttaccagtttaatgggtct
KLF6 F tgtagtattttttaggagttttagat 375 53.7 56.5
R aacaaaacaaattaatccccataac
LBC F tagttttttttagtaatttgagtgt 449 54.3 57.9
R cacaaatcacaaaacttcccccaac
MAFA F ttgttatggggataagtaaatgagg 407 54.2 57.0
R aaaaaaaaaactacaaacctctccc
MAL F gggtttgggattaatgtagt 348 52.8 53.8
R ctaaccaatcaaaaacctaat
MSL-1 F gggatttttattattatttttgtt 346 53.9 57.0
R aaataccttaacctcctacaaccc
NKX2-1 F ggggttatatttgaagtttgg 442 57.4 54.1
R aaaacatcacaaccccaacac
NKX3-1 F agttaattggattgtttgttttgat 608 56.0 57.3
R tccctctaactctaactctaactcc
NKX3-2 F gtttttaaagatttagaggaggaagt 329 56.0 57.5
R ccaacccaatctaatacacattaac
NOG F ctcaaatacctctactatcaactc 454 62.0à47.0 57.5
R gttttgtattttgagtggtttagtt
PCDHB7 F ggggtaaaatgaggatttttttt 436 56.0 54.8
R aaaactctctaaaaccacacaattc
PPM1L F ttttgatagttgggtgggaatattt 317 61.0 60.2
R aaaattccaattccttcacacacc
PSMD10 F tagggagggggagggtagttatag 362 59.8 52.5
R ctaccctcacaaacttaacccctaa
PTPRT F agtttgtttttgaggttgtagttgt 505 56.0 56.4
R actaaaaacctatcctccccactaa
RPL35 F gtttagattgggagatttgtaag 407 58.9 56.8
R caactctcaccattactacacaa
SALL1 F tattttaaaagaaaagatttaagaagttaa 458 54.0 56.2
R caccatctccctcaaactaaaatac
SATB1 F aggaggaagaagaggtatttatag 429 67.0à55.0 55.6
R acaaaactaatcaaaaatttccc
SFRP2 F agattatgaaggaggtgttggagta 440 58.5 54.9
R gggtagaagttttgtttgatttggt
SHARPIN F gttgggttttttagttaaggtaagag 386 56.0 56.7
R taacccaaccaaacaaataaatatc
SIGIRR F taaggtaggttttttggaggaaag 367 59.3 57.5
R cacctactaccaaaacctaaaactc
SNX4 F ggtttagaaggagtaggttagatta 542 59.8 58.3
R accacacaaaccataaaaactctac
SOX4 F gaggagttgaggggttagtt 451 54.2 55.6
R ctatctttaaacaacttccaac
SOX9 F tttattaaagagattttgggttgg 424 52.7 56.0
R ataaaaaaatccaaaaaattcatac
SSRP1 F gtttattttttttgtggtttgtgta 361 55.0 56.3
R ccaactcaaaatacaaaatttccct
SRF F gttataggggtaggaaagtgag 647 58.0 59.2
R ataaactccatcttaatcttcac
ML-F gggagtgtcgggttgagtc 116 56.0
ML-R ccccaactta aatcg/aataacataaca
ML-Probe 6FAM-aagtcgatggcggcggtt-TAMRA
RT-PCR-F tgctgaatgccttctccca 166 56.0
RT-PCR-R gcctgctgccctatcaca
SRFBP-1 F agttattagagagaaaaattaaggt 471 56.0 56.8
R tttaaaaacccttaaaatcaac
SFRS2B F tgggttttggagtatttggt 257 53.0 56.2
R ccaccttaaaaataatcatacc
TBX5 F gtttttggtatttgggagtaaata 484 57.0 55.4
R aaaaaaaactaaacctacccaacac
TEGT F aagtttaggtttttgaattaagtag 343 51.7 55.9
R tccttcaaaataccatctatcc
TFPI2 F aggtaggtttaattttttaatttgg 344 54.0 55.7
R tacctattaactcctaaacaacatc
RT-PCR-F gcgatgctgctcaggag 114 60.0
RT-PCR-R tctgcgtgtacctgtcgtagtag
TIGD7 F gaagttgttagttttgaggtttaat 342 56.0 57.9
R caaaaaaacaaccattttcc
TIMP3 F ggtattattttttataaggatttg 553 56.0 56.3
R caatcccccaaactccaact
WDR32 F agtggtatgttatttattgtgtgtgg 384 59.7 57.0
R atacccctattcaactaccacttc
ZBTB7A F tggttgtattgtatagttattgtagggg 315 56.0 58.0
R ccccttcctctaaaacttaaaaaaa
ZNF382 F ggggaggttagggaggtaatta 437 57.0 56.6
R cacactcaaaaacaatctcaaatac
ML-F gatcgcggtatttagacg 96 58.0
ML-R ttcgcgaaactcgctccg
ML-Probe 6FAM-gcccgaaacccgactcg-TAMRA
RT-PCR-F attgccacttcgtatctg 173 58.0
RT-PCR-R atgcctgtcttggtattctt
ZNF586 F tggttgagttaatgaagttttagagaag 406 56.0 55.4
R taacaaaaacctaaactacaaaaac
ZNF775 F gttagtgggtgagttatgggtatag 421 56.0 54.5
R ctaccttataaaaaactaaaaaccc
a F/R: primer set for DHPLC and bisulfite sequencing; ML-F/R/Probe: primer set and probe for MethyLight assay; RT-PCR-F/R: primer set for quantitative RT-PCR
Quantitative reverse transcription PCR (qRT-PCR)
Total RNA was isolated using TRIzol (Invitrogen, Carlsbad, CA, USA) and reverse transcribed with MPMLV (Promega, Madison, Wisconsin, USA). Quantitative RT-PCR was carried out in triplicate for the target genes using power SYBR Green PCR Master Mix (P/N 4367659, Applied Biosystems, Carlsbad, CA, USA). The primer and PCR annealing temperature for the tested genes are listed above. GAPDH mRNA was used as the reference control.
Bisulfite modification
Bisulfite conversion was performed by adding 5M sodium bisulfite to 2 mg DNA samples (18).
Clone sequencing
PCR products of the CGIs amplified with the CpG-free universal primer sets were cloned with the pGEM-T Easy kit (Promega, Madison, USA) and sequenced with Applied Biosystems 3730xl DNA Analyzer at SinoGeneMox Company (Beijing, China).
Tissue microarray (TMA)
TMA sections containing the paired intestinal type GC and SM (n=48) were used to determine the protein expression of target genes in IHC assays as described previously (22).
Immunohistochemical staining (IHC)
Mouse anti-human GFRA1 monoclonal antibody (1:100; Santa Cruz, sc-271546), rabbit anti-human ZNF382 polyclonal antibody (1:20; Novus Biologicals, NBP2-13569), and Rabbit anti-human SRF polyclonal antibody (Abcam, Ab53147) were used as primary antibodies in the IHC analysis, combined with HistostainTM-Plus Kit (ZYMED, SP-9000) and DAB Kit according to the manufacture’s instructions. Mouse or rabbit IgG (Beijing Zhongshan Golden Bridge Biotech, ZDR-5006 or ZDR-5003) was used as the corresponding negative control. The slide was counterstained with hematoxylin.