Supplementary Figure 1. 1,000-cell Scale Nano-ChIPseq Validation Comparison between small-scale ChIPseq (ie Nano-ChIPseq) and Standard ChIPseq for

Transcription

1 Supplementary Figure 1. 1,000-cell Scale Nano-ChIPseq Validation Comparison between small-scale ChIPseq (ie Nano-ChIPseq) and Standard ChIPseq for K27me3 in murine ES cells. A representative Genome Browser track is shown. Additional details are presented in Ng et al., (2013) Developmental Cell 1.

2 Supplementary Figure 2. Nano-ChIPseq Peak Calling and Peak Merging. (a) Representative UCSC browser view of Nano-ChIPseq tag density for 5 histone modifications in patient normal ( N). Peak regions are indicated above each ChIPseq track. The gene track shows RefSeq transcripts. (b) K4me3 peak profile of 6 tissue samples. Merged peak regions are indicated below the RefSeq transcript track.

3 Supplementary Figure 3. Identification and Removal of Amplified Regions. Representative views of amplified loci around the KRAS locus in tissue C (a) and MYC in tissue C (b). Regions exhibiting an abundance of sequence tags in the cancer input DNAs were removed from subsequent analysis.

4 Supplementary Figure 4. Overlap of Chromatin Marks in GC/Normal Pairs. Overlap of chromatin mark regions in normal (top) and cancer tissue (bottom) for sample pairs (a) and (b). Numbers represent the fraction of 1 st set regions (vertical) overlapping with the 2 nd set regions (horizontal). Blue = exclusive, Red = overlapping.

5 Supplementary Figure 5. Null distributions of overlap between enhancer predictions and P300/CEBP/CTCF binding sites in HeLaS3. The overlap of predicted enhancers with each different transcription factor binding site repertoire is statistically significant (P<10-4 ). Similar results were obtained for K562 cells.

6 Supplementary Figure 6. Nano-ChIPseq RPKM Pre-processing. (a-d) Boxplots of log2-transformed ChIPseq RPKM values (after ComBat normalization) for promoter and predicted enhancer regions. (e-h) Raw p-value distributions from linear model fitting.

7 Supplementary Figure 7. Validation of Nano-ChIPseq altered chromatin peaks by qpcr Using qpcr we validated 18 altered chromatin peaks identified by Nano-ChIPseq analysis as being different between tumor and normal samples (9 K27ac, 9 K4me3; corresponding to 14 genomic regions). log 2 fold change<0 is defined as Loss, log 2 fold change>0 is defined as Gain. All 18 peaks showed a similar concordance between the mean Nano-ChIPseq fold change and mean qpcr fold change. qpcr was performed on 3 GC/normal pairs for which tissue was available.

8 Supplementary Figure 8. PCA plots of GCs and Normal Tissues. (a,b) and (d,e) PCA plot of GCs (purple) and normal (blue) tissues using (a) all identified K4me3 (promoter) and (b) associated K27ac (activity); or (d) all identified K4me1 (predicted enhancer) and (e) associated K27ac regions. (c and f) PCA plot using K27ac regions exhibiting somatic alterations.

9 Supplementary Figure 9. Cryptic promoter usage in GC cell lines (a) Venn diagram of K4me3 and K27ac Nano-ChIPseq overlap with cryptic promoters in GC cell lines (e.g. N87, YCC3, OCUM1 and SNU16). Of 368 cryptic promoters identified, 157 are found in N87, 91 are found in YCC3, 187 are found in OCUM1 and 197 are found in SNU16. When combined, a total set of 249 (67.7%) cryptic promoters were observed in the cell lines. (b) Example of a cryptic promoter region (orange box) marked by both K4me3 (top brown) and K27ac (bottom purple) in GC cell lines (N87, YCC3) and tumor sample , but not in matched normal gastric tissue.

10 Supplementary Figure 10. RT-qPCR Validation of Non-RefSeq Transcripts. RT-qPCR validation results for 10 non-refseq transcripts in GCs and normal tissues, compared to FPKM values derived from RNAseq analysis. Primer sets are provided in Supplementary Table 10.

11

12 Supplementary Figure 11. MET Gene RNAseq Alignment. (a) RNAseq tag alignments supporting exon-intron structures of expressed RNAs at the MET gene locus. MET RefSeq transcripts are indicated in the top annotation. (b,c) Close-up view of RNAseq alignments. Exon-intron boundaries for cryptic promoter-driven exons were confirmed by manual inspection of sequence tag alignments. (d) Visualization of MET cryptic promoter showing location of representative RNAseq reads and K4me3 enrichment. The downstream MET RefSeq exon is shown. (e) 5 RACE analysis. Close-up cryptic promoter view with RNAseq and K4me3-enriched reads. The location of the 5 RACE primer is indicated, and the product of the 5 RACE product from GC Hs746T cells is shown*. (f) Schematic of predicted Met protein isoforms driven by RefSeq or cryptic promoters. (g) Western blotting. Full length Met and predicted isoforms were monitored using antibodies targeting the Met C-terminal cytoplasmic domain. The band size of the predicted isoform corresponding to the truncated Met variant driven by the internal cryptic promoter is 978 aa (~100 kda), while the RefSeq associated full length Met receptor is 190kDa 2. β- actin was used as a loading control. NUGC4 cells are predicted to express both the truncated Met variant and full-length Met, while KATOIII cells are predicted to predominantly express full length Met. *Hs746T cells were not used for Western blotting as these cells contain an exon 14 Met splice site mutation that also leads to other aberrant Met proteins 3.

13 Supplementary Figure 12. MET RNAseq Analysis and Full Image of Met Western blot. (a) RNAseq analysis demonstrating expression of exons associated with the MET internal cryptic promoter (red box) in GC C and NUGC4 cells (red arrows) but not in N (matched normal gastric tissue) and KATOIII cells (b) Original Met and β-actin Western blot image for NUGC4 and KATOIII as shown in Supplementary Fig. 11g. Molecular weight markers are indicated.

14

15 Supplementary Figure 13. NKX6-3 RNAseq Alignment. (a) RNAseq tag alignments supporting exon-intron structures of expressed RNAs. NKX6-3 RefSeq transcript (NM152568) is shown in green, and predicted cryptic-promoter expressed mrnas are indicated at the top. (b-f) Close-up view of RNAseq alignments. Exon-intron boundaries were confirmed by manual inspection of sequence tag alignments. (g) Gel photo of two distinct 5 RACE products from 7 GC cell-lines. (h) Genome browser view showing positions of 5 RACE fragments from NUGC3 (larger RACE product) and KATOIII (smaller RACE product) cell-lines. The location of the 5 RACE primer is indicated. Both products validate the expression of a 5 non-refseq exon, where NUGC3 has a larger product. Transcript 5 ends are shown by the red arrows (i) Predicted mrna and polypeptide structures. Location of the NKX6-3 homeodomain is indicated based on the RefSeq database.

16 Supplementary Figure 14. HOXB9 Locus RNAseq Alignment. (a) RNAseq alignments at the HOXB9 locus. Multiple spliced RNA isoforms were predicted based on RNAseq alignments. (b-h) Exon-intron boundaries were confirmed by RNAseq tag alignments. (i) Coding potential of predicted mrnas and microrna precursors are indicated. The oncogenic mirna MIR196A also falls in this region.

17 Supplementary Figure 15. Microarray Validation Cohort. Heatmap of log2-transformed expression of upregulated K4me3-marked cancer-associated genes (n =218) in independent microarray data. Cancer (n = 185) and Normal (n = 89) samples are from Singapore. Cancer and Normal samples show distinct patterns of expression where the majority of K4me3-marked cancer-associated genes are upregulated in tumors.

18 Supplementary Figure 16. Association of Cancer-associated Genes with Clinicopathologic Features. Mosaic plots of factors with significant association with K4me3-marked cancer-associated genes: (a) M- stage, (b) Lauren s histopathology, and (c) Intrinsic signature from Tan et al. 4. Higher M-stage (p = 0.033, Pearson s Chi-square test), diffuse Lauren s histopathology (p = 9.99x10-5, Pearson s Chi-square test), and GDIFF intrinsic signature classification (p =1.46x10-11, Pearson s Chi-square test) are significantly associated with high-expression of the K4me3-marked cancer-associated genes. Red: high-enrichment of signature; blue: low-enrichment of signature.

19 Supplementary Figure 17. Overlap of GC Promoters and Enhancers with ENCODE Data. (a) Frequency of TFBS in promoter regions. (b) Frequency of TFBS in predicted enhancer regions.

20 Supplementary Figure 18. Validation of somatic mutations by Sanger sequencing. (a-c) Three representative Sanger traces are shown, depicting somatic mutations. Mutation sites are indicated by arrows. C and N refer to Cancer and Normal respectively.

21 Supplementary Figure 19. CDH10 Locus Somatic Mutation Analysis. (a) Genome browser view of CDH10 locus. (b) Magnified view. (c) K4me3 ChIPseq tag alignment, showing variant and allele bias. Note that the mutation is a dinucleotide substitution compared to the hg19 reference. (d) Sanger sequencing traces of normal and cancer input DNA and K4me3 ChIP DNA. Cancer input contains a small fraction of the mutant allele, and the mutant allele is enriched by K4me3 ChIP.

22 Supplementary Figure 20. HOXA5 Locus Somatic Mutation Analysis. (a) Genome browser view of HOXA5 locus. (b) K4me3 ChIPseq tag alignment, showing variant and allele bias. (c) Sanger sequencing traces of normal and cancer input DNA and K4me3 ChIP DNA. Cancer input contains a small fraction of mutant allele, and the mutant allele is enriched by K4me3 ChIP.

23 Supplementary Figure 21. FAR2 Locus Somatic Mutation Analysis. (a) Genome browser view of FAR2 locus. (b) Magnified view. (c) K4me3 ChIPseq tag alignment, showing variant and allele bias. (d) Sanger sequencing traces of normal and cancer input DNA and K4me3 ChIP DNA. Cancer input contains a small fraction of mutant allele, and the mutant allele is enriched by K4me3 ChIP.

24 Supplementary Table 1. Clinical Data for Samples Analyzed by Nano-ChIPseq and RNAseq. Tissue ID Age at Surgery Gender site.of.tumour StageT.7 StageN.7 StageM.7 AJCC7 Grade H.pylori.status moderately male lesser curve T4a N3a m1 4 differentiated yes poorly male lesser curve T4a T3b m1 4 differentiated yes poorly female PYLORUS T4a T3b m1 4 differentiated UNKNOWN poorly female incisura T4a T3b m0 3C differentiated UNKNOWN Lauren's Classification "intestinal type" adenocarcinoma "diffuse type" adenocarcinoma "diffuse type" adenocarcinoma "intestinal type" adenocarcinoma male unknown T2 N1 m0 2A undifferentiated UNKNOWN mixed/others ChIPseq male lesser curve T3 N3a m0 3B poorly differentiated yes "intestinal type" adenocarcinoma RNAseq male antrum T2 N1 m0 2A well differentiated UNKNOWN "intestinal type" adenocarcinoma RNAseq female lesser curve T2 N1 m0 2A moderately differentiated no "intestinal type" adenocarcinoma RNAseq male CARDIA T2 N1 m0 2A poorly differentiated UNKNOWN "intestinal type" adenocarcinoma RNAseq male lesser curve T4a T3b m0 3C poorly differentiated yes "diffuse type" adenocarcinoma RNAseq male antrum T4a N3a m1 4 moderately differentiated UNKNOWN "intestinal type" adenocarcinoma RNAseq female greater curve T1b N0 m0 1A moderately differentiated no "intestinal type" adenocarcinoma RNAseq male antrum T2 N1 m0 2A moderately differentiated UNKNOWN "intestinal type" adenocarcinoma RNAseq Platform ChIPseq ChIPseq ChIPseq ChIPseq

25 Supplementary Table 2. ChIPseq Library and Peak Call Summary. Library Patient Tissue ChIP antibody Read length Uniquely mapped read CCAT3 fold-cutoff filtered CCAT Peak region CHG Cancer Input ,569,842 CHG Normal Input ,367,968 CHG Cancer Input ,306,207 CHG Normal Input ,360,435 CHG Cancer Input ,686,506 CHG Normal Input ,097,151 CHG Cancer H3K4me ,268, ,652 CHG Normal H3K4me ,235, ,336 CHG Cancer H3K4me ,437, ,339 CHG Normal H3K4me ,563, ,361 CHG Cancer H3K4me ,614, ,158 CHG Normal H3K4me ,395, ,861 CHG Cancer H3K27ac ,047, ,580 CHG Normal H3K27ac ,024, ,042 CHG Cancer H3K27ac ,004, ,222 CHG Normal H3K27ac ,336, ,208 CHG Cancer H3K27ac ,575, ,955 CHG Normal H3K27ac ,176, ,619 CHG Cancer H3K4me ,374, ,934 CHG Normal H3K4me ,100, ,932 CHG Cancer H3K4me ,464, ,087 CHG Normal H3K4me ,970, ,379 CHG Cancer H3K4me ,591, ,963 CHG Normal H3K4me ,976, ,074 CHG Cancer H3K36me ,870, ,308 CHG Normal H3K36me ,978, ,980 CHG Cancer H3K36me ,758, ,449 CHG Normal H3K36me ,309, ,879 CHG Cancer H3K36me ,619, ,912 CHG Normal H3K36me ,690, ,370 CHG Cancer H3K27me ,833, ,363 CHG Normal H3K27me ,631, ,910 CHG Cancer H3K27me ,335, ,308 CHG Normal H3K27me ,850, ,862 CHG Cancer H3K27me ,847, ,694 CHG Normal H3K27me ,898, ,682 CHG Normal Input ,653,370 CHG Cancer Input ,916,056 CHG Normal Input ,021,674 CHG Cancer Input ,781,611 CHG Normal H3K27ac ,671,862 CHG Cancer H3K27ac ,597,536 CHG Normal H3K27ac ,983,068 CHG Cancer H3K27ac ,051,266 CHG Normal H3K4me ,841, ,644 CHG Cancer H3K4me ,794, ,997 CHG Normal H3K4me ,908, ,131 CHG Cancer H3K4me ,713, ,382 CHG Normal H3K4me ,451, ,116 CHG Cancer H3K4me ,926, ,974 CHG Normal H3K4me ,946, ,007 CHG Cancer H3K4me ,434, ,167 CCAT peak call results were filtered by fold-above background cut-off to obtain peak regions.

26 Supplementary Table 3. Accuracy of Enhancer Predictions Using ENCODE ChIPseq data and CCAT version 3.0 (FDR < 0.05 and excluding H3K4me3 enriched regions). Cell Line Transcription Factor Total Enhancer Predictions % TFBS overlap w/ Enhancer Prediction Sensitivity Specificity 52% 67% 51% HeLaS3 P300 HeLaS3 CEBP 40% 65% 53% 73,929 HeLaS3 CTCF 23% 55% 50% HeLaS3 P300, CEBP, CTCF 24% 63% 54% K562 P300 30% 37% 39% K562 CEBP 29% 50% 40% 109,082 K562 CTCF 25% 53% 40% K562 P300,CEBP, CTCF 23% 43% 35%

27 Supplementary Table 4. Cryptic Promoters in Primary Gastric Adenocarcinomas. Transcript Database Cryptic Promoters Overlap with Annotated TSS RefSeq* Ensembl74** Gencodev * Values provided in Table correspond to RefSeq transcripts as of Nov The updated RefSeq database (Release 63) has 22 additional promoter regions associated with annotated transcripts ** Ensembl 74 contains Met and NKX6-3 isoforms described in Fig. 2 of the Main Text

28 Supplementary Table 5. Clinical Characteristics of Validation Cohort. Age (years) range mean ± standard deviation Singapore GC Cohort (n = 185) (1 missing) 64.5 ± 12.9 (1 missing) Gender (percent) Female 68 (36.8) Male 116 (62.7) missing 1 (0.54) Stage (percent) 1 29 (15.7) 2 30 (16.2) 3 66 (35.7) 4 59 (31.9) missing 1 (0.54) Lauren's histopathology (percent) Intestinal 92 (49.7) Diffuse 72 (38.9) Mixed/ unclassifiable 20 (10.8) missing 1 (0.54) Intrinsic signature** (percent) GINT 92 (49.7) GDIFF 70 (37.8) Ambiguous 23 (12.4) Helicobacter Pylori status (percent) Positive 59 (31.9) Negative 37 (20.0) missing 89 (48.1) Median overall survival* (months) 22.5 (1 missing) Number of overall death events 110 (2 missing) *Defined as time from surgery to last follow-up **Classified according to Tan et al. (2011)

29 Supplementary Table 6. Association of Clinicopathological Factors with K4me3-marked Cancer - Associated Genes. Clinicopathological factor class of factor Pearson's Chi-sq P- value t-test P-value Direction of association Age continuous N.A N.A. Gender categorical N.A. N.A. Site of tumor categorical N.A. N.A. Grade categorical N.A. N.A. TMN stage (AJCC7) categorical N.A. N.A. T.stage categorical N.A. N.A. M.stage categorical N.A. positive Number of lymph nodes continuous N.A N.A. Helicobacter Pylori status categorical N.A. N.A. Lauren's histopathology categorical 9.99E-05 N.A. positive Intrinsic signature* categorical 1.46E-11 N.A. positive Ulcerative status categorical N.A. N.A. Adjuvant 5FU therapy categorical N.A. N.A. *Intrinsic signature from Tan et al. (2011)

30 Supplementary Table 7. Transcription Factor Binding Site Overlap Analysis. Yellow: p<0.05 for promoter regions. Blue: p<0.05 for predicted enhancer regions. TFs all_k 4me3 all_k 4me1 gainp romot er losspr omote r gaine nhanc er losse nhanc er all_k 4me3 _per1 0kb gainp romot er_pe r10kb losspr omote r_per 10kb all_k 4me1 _per1 0kb gaine nhanc er_pe r10kb losse nhanc er_pe r10kb Pval_H4 me3_ga in corpval _H4me3 _Gain Pval_H4 me1_ga in corpval _H4me1 _Gain ARID3A E E E E-36 ATF E E E E-12 ATF E E E E-02 ATF E E E E-40 BACH E E E E-04 BATF E E E E-05 BCL11A E E E E-03 BCL E E E E-17 BCLAF E E E E-03 BDP E E E E+00 BHLHE E E E E-12 BRCA E E E E-13 BRF E E E E+00 BRF E E E E+00 CBX E E E E-08 CCNT E E E E-06 CEBPB E E E E-54 CEBPD E E E E-15 CHD E E E E+00 CHD E E E E-31 CREB E E E E-16 CTBP E E E E+00 CTCF E E E E-11 CTCFL E E E E+00 E2F E E E E-09 E2F E E E E-11 E2F E E E E-05 EBF E E E E-03 EGR E E E E-01 ELF E E E E-17 ELK E E E E-04 ELK E E E E-10 EP E E E E-71 ESR E E E E-21 ESRRA E E E E-01 ETS E E E E-12 EZH E E E E+00 FAM48A E E E E-04 FOS E E E E-72 FOSL E E E E-19 FOSL E E E E-80 FOXA E E E E-32

31 FOXA E E E E-23 FOXM E E E E-01 FOXP E E E E-03 GABPA E E E E-40 GATA E E E E-03 GATA E E E E-19 GATA E E E E-11 GRp E E E E+00 GTF2B E E E E+00 GTF2F E E E E-19 GTF3C E E E E+00 HDAC E E E E+00 HDAC E E E E-22 HDAC E E E E+00 HDAC E E E E+00 HMGN E E E E-03 HNF4A E E E E-20 HNF4G E E E E-22 HSF E E E E+00 IKZF E E E E+00 IRF E E E E-02 IRF E E E E-01 IRF E E E E+00 JUN E E E E-68 JUNB E E E E-23 JUND E E E E-59 KAP E E E E-14 KDM5A E E E E+00 KDM5B E E E E+00 MAFF E E E E-13 MAFK E E E E-14 MAX E E E E-36 MAZ E E E E-19 MBD E E E E-11 MEF2A E E E E-01 MEF2C E E E E+00 MTA E E E E+00 MXI E E E E-12 MYBL E E E E-23 MYC E E E E-48 NANOG E E E E+00 NFATC E E E E+00 NFE E E E E-01 NFIC E E E E-20 NFYA E E E E-08 NFYB E E E E-05 NR2C E E E E-02

32 NR2F E E E E-11 NR3C E E E E-55 NRF E E E E-01 PAX E E E E-03 PBX E E E E+00 PHF E E E E-01 PML E E E E-03 POLR2A E E E E-44 POLR3G E E E E+00 POU2F E E E E-02 POU5F E E E E+00 PPARGC1 A E E E E-02 PRDM E E E E-08 RAD E E E E-21 RBBP E E E E-03 RCOR E E E E-30 RDBP E E E E+00 RELA E E E E-03 REST E E E E-27 RFX E E E E-35 RPC E E E E+00 RUNX E E E E-01 RXRA E E E E-25 SAP E E E E+00 SETDB E E E E-02 SIN3A E E E E+00 SIN3AK E E E E-19 SIRT E E E E-02 SIX E E E E-07 SMARCA E E E E-05 SMARCB E E E E-16 SMARCC E E E E-35 SMARCC E E E E-05 SMC E E E E-22 SP E E E E-27 SP E E E E+00 SP E E E E+00 SPI E E E E+00 SREBP E E E E+00 SRF E E E E-04 STAT E E E E-26 STAT E E E E-02 STAT E E E E-87 STAT5A E E E E-06 SUZ E E E E+00 TAF E E E E-24

33 TAF E E E E+00 TAL E E E E-08 TBL1XR E E E E-06 TBP E E E E-34 TCF E E E E-42 TCF E E E E+00 TCF7L E E E E-63 TEAD E E E E-25 TFAP2A E E E E-27 TFAP2C E E E E-35 THAP E E E E+00 TRIM E E E E-08 UBTF E E E E+00 USF E E E E-21 USF E E E E-16 WRNIP E E E E+00 YY E E E E-21 ZBTB E E E E-40 ZBTB7A E E E E-08 ZEB E E E E+00 ZKSCAN E E E E-05 ZNF E E E E-05 ZNF E E E E-18 ZNF E E E E-01 ZNF E E E E+00 ZZZ E E E E+00

34 Supplementary Table 8. Genetic Variation in Nano-ChIPseq Data. Germline Variants Somatically altered Detected elements Somatic Mutations Somatically altered Detected elements Tissue Gain Loss Gain Loss , , , , , Total 439,160 1, Germline SNVs and somatic mutations in 5 tumor/normal pairs detected by MuTect at minimum 20x coverage and/or validated using Sanger sequencing in both tumor and normal. Sequence coverage for the last two pairs ( and ) was lower than the first three, hence the lower rate of SNV identification. 439,160 dbsnp (ie germline) SNVs were detected across the 5 patients (249,786 unique SNPs). Approximately 188,500 were heterozygous in at least one sample. Across the 5 patients, 1,760 SNPs mapped to somatically altered elements. 947 SNPs were unique, of which 714 were heterozygous. Similarly, 144 somatic mutations were detected across the 5 patients. Sanger sequencing confirmed the somatic mutation status of 55 out of 71 sites (77%).

35 Supplementary Table 9. Assessing Allele Bias at 16 Sites Using Input DNAs and ChIP-enriched Reads. 11 sites were validated using quantitative PyroMark sequencing (5 sites were not validated due to PCR or Pyrosequencing (PSQ) failure highlighted in grey). 8 sites (73%) confirm allele bias (>0.30, in bold) in ChIP-enriched reads. Tissue Region Coordinate (0-based) Ref SNP Alternate Allele Fraction by PyroMark INPUT K4me3 K27ac Tumor Normal Bias Tumor Normal Bias Tumor Normal Bias Regulome DB Score Nearby genes Gain chr2: T C b ID Gain chr2: C G b CAPN Loss chr4: T C a SORBS Gain chr6: A G f AK Gain chr6: C PCR failure 1a ZDHHC Gain chr7: C PSQ failure 1f CLDN Gain chr7: G PSQ failure 2b CLDN Gain chr7: T G b CFTR Gain chr8: T C b FER1L Gain chr9: T A b MIR31HG, MTAP Gain chr9: G C b MIR31HG, MTAP Gain chr11: C PCR failure 2b KCNE Gain chr15: G PCR RPUSD2, 2c failure CASC Gain chr18: G A b SERPINB Gain chr19: A G f KLK Gain chr20: T C b AK307192

36 Supplementary Table 10. PCR, qpcr, 5 RACE, PyroMark and Cloning Primers Description Non-RefSeq exon expression Exon1, chr1: Forward Exon1, chr1: Reverse Exon2, chr1: Forward Exon2, chr1: Reverse Exon3, chr7: Forward Exon3, chr7: Reverse Exon4, chr8: Forward Exon4, chr8: Reverse Exon5, chr8: Forward Exon5, chr8: Reverse Exon6, chr8: Forward Exon6, chr8: Reverse Exon7, chr11: Forward Exon7, chr11: Reverse Exon8, chr17: Forward Exon8, chr17: Reverse Exon9, chr19: Forward Exon9, chr19: Reverse Exon10, chr19: Forward Exon10, chr19: Reverse 5' RACE primers 1st strand MET Refseq exon 3 1st strand NKX6-3Refseq exon 1 PCR MET exon 3 PCR NKX6-3 exon 1 PCR NKX6-3 exon 1 for nested PCR qpcr and Pyrosequencing TNK2_A45_R_5btn TNK2_A45_F TNK2_A45_PSQ_F NUDT4_SN78_R_5bio NUDT4_SN78_F NUDT4_SN78_PSQ_F KLK1_E7_F436_btn KLK1_E7_R577 KLK1_E7_PSQ_R HOXA11_A64_R_5btn_2 HOXA11_A64_F HOXA11_A64_PSQ_F2 Reporter cloning FOS-99_BglII FOS-99_HindIII HOX11AS_F308_BglII HOX11AS_R645_BglII Differential promoters Promoter1, chr1: , Forward Promoter1, chr1: , Reverse Promoter2, chr1: , Forward Promoter2, chr1: , Reverse Promoter3, chr2: , Forward Promoter3, chr2: , Reverse Sequence (5'>3') AGGCAGGAGAGTAGAGTCTGGAGG GTTCTAGGAAGTCAGCGTGAAACG TGGCGGACTCAACAAGGC GGAAGGCAGGAAGTGGCT ACTTTGGCAAGGTTAGGT GTTGTAGGGATGGGTTCT CCTGCGACTCGGTCATGC ACACCCGGCCCACCTTCA GTTGTTCAGCAGGAACGT TTTGGGGCTAGAGAGTGG GATTTTGCTGTGTTCCTA CTTCTCCATTCTTCCTCT GGACCTGCCACCTCCAAAC TCCTGAAAGAGCTGCCTTC AGGTGCCCCCAGGTGACTC GCCCTTTCGCTGCTATGCC ATGCTCCCCCTTTTCCACC AATGTCCCTTCTCCCCTTG TCCCAGCCTGCATTCCAT GGTTTGCCTCGGGTTCTT CTTCAGTGCAGGG GAAGGTAGGCTCCTC GGCTCCAGGGTCTTCACCTCCA CCAGGCTGAGCACCGAGAAGGC GCTTGCGCAGCAGCAGGCGGAT [Btn]TGGGACCCCTAGGGAGAAGA CAGAGGCCGGTGCTGAGA TCTGGAAATCTCAGCAC [Btn]GCCAAGACTGGGCCATGT CCTACGAACAGAAATAAAACAATTGTGA TGGGAGAAGCAGAGTG [Btn]TTGAGAGTGCAAAAGGCAAATG GCTGTCCCTGTCTTTTATCCTTCT CCTCCCCTCCGCCTT [Btn]CCCTTTTTCCCCTAGATTTGGT CCCAAGGCTGCGCAATAT TGAATGTGAAGGTTTCTC GTAGCTGCATAGATCTGCGCGCCACCCCTCTGGCGCCACCGT GTAGCTGCATCAAGCTTGCCGGCTCAGTCTTGGCTTCTC AGCTATGCTGAGATCTGAAGATGGATGAGGGAAAAGGTT AGCTATGCTGAGATCTTGTTGGACCTCCTGCAAAGC ACCTGCGACTGTCTCTTT GGCTGTTTACCGTTGTTT CTTTGTTCTCCTCTGCTT CTGTATTTGTCCCGATTG AGGGTGTGAATGATGGTA TGTCTCCTCGTATGCTGC

37 Promoter4, chr4: , Forward Promoter4, chr4: , Reverse Promoter5, chr5: , Forward Promoter5, chr5: , Reverse Promoter6, chr5: , Forward Promoter6, chr5: , Reverse Promoter7, chr7: , Forward Promoter7, chr7: , Reverse Promoter8, chr8: , Forward Promoter8, chr8: , Reverse Promoter9, chr9: , Forward Promoter9, chr9: , Reverse Promoter10, chr11: , Forward Promoter10, chr11: , Reverse Promoter11, chr12: , Forward Promoter11, chr12: , Reverse Promoter12, chr18: , Forward Promoter12, chr18: , Reverse Promoter13, chr18: , Forward Promoter13, chr18: , Reverse Promoter14, chr20: , Forward Promoter14, chr20: , Reverse TTCAGCAAAGGAGAAACA GAGTGAGAGTGGCAAAAC ATGACTTCTGGGTTTCTG CAACAGACATCATCGTGG ATAGAGATTGGCAAACCG TGTCTCCTCGTATGCTGC AAGCACTTATCGCCAATC ATACATCCCTGACTCCAC AAAACCTGATATGTCCCT TAGTGCCAACCTCTAACC CCCACATACCCATTACTT AGCAGAAACTTCAACACA TTCTTTCCTGAATCCTGC GACTCAGGGAGAGGAGAG TGTGGCTAAGAGGTTGGT AGGGAGCCTGCGATAAGT CACCTTTACCTCCCAACA ATTCCTTTCTCTTGTTCC AACAAGAAAGCAGGTGAT GGCTCCCTCTACACACAG ATCCCACCATTAGAGAGA CGCCATCAGATCAGAATC

38 Supplementary References 1. Ng JH, et al. In vivo epigenomic profiling of germ cells reveals germ cell molecular signatures. Dev Cell 24, (2013). 2. Rodrigues GA, Naujokas MA, Park M. Alternative splicing generates isoforms of the met receptor tyrosine kinase which undergo differential processing. Mol Cell Biol 11, (1991). 3. Asaoka Y, et al. Gastric cancer cell line Hs746T harbors a splice site mutation of c-met causing juxtamembrane domain deletion. Biochem Biophys Res Commun 394, (2010). 4. Tan IB, et al. Intrinsic subtypes of gastric cancer, based on gene expression pattern, predict survival and respond differently to chemotherapy. Gastroenterology 141, , 485 e (2011).