Supplementary Table S1

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RFX4 (320). AP3B1 (28,984). Downstream gene. -. +. +. Strand. 50,274,763. 105,500,640. 77,304,600. Start. 50,275,039. 105,500,841. 77,304,921. End.
Supplementary Table S1

HCT-116

HCT-116AZA

Geometric mean ratio

CpG island

HCT-116 methylation

Normal tissue methylation

Uc.399+A

22.63

516.13

22.81

No

-

-

Uc.230+

22.63

86.68

3.83

No

-

-

Uc.282+A

488.13

1252.55

2.57

Yes

Yes

Yes

Uc.283+A

3415.75

7780.97

2.28

Yes

Yes

No

Uc.278+

340.79

574.47

1.69

No

-

-

Uc.3+

704.08

1091.92

1.55

No

-

-

Uc.196+

311.11

479.65

1.54

No

-

-

Uc.160+

400.81

595.83

1.49

Yes

Yes

No

Uc.469+A

2593.86

3802.13

1.47

Yes

Yes

Yes

Uc.392+A

607.84

827.49

1.36

Yes

No

-

Uc.34+A

1275.43

1693.67

1.33

No

-

-

Uc.346+

371.58

486.92

1.31

Yes

Yes

No

Uc.468+

1969.86

2451.82

1.24

No

-

-

Uc.117+

2666.29

3283.17

1.23

No

-

-

Name

Supplementary Table 1. Summary of the T-UCR microarray data. The geometric mean of intensities, ratio of geometric means, presence of CpG island and methylation status are represented.

Supplementary Table 2. Primer sequences

Primers for bisulfite genomic sequencing: BS-160+-s Sense BS-160+-as Antisense BS-283+A-s Sense BS-283+A-as Antisense BS-283+A2-s Sense BS-283+A2-as Antisense BS-346+-s Sense BS-346+-as Antisense BS-346+2-s Sense BS-346+2-as Antisense BS-282+A3-s Sense BS-282+A3-as Antisense BS-392+A-s Sense BS-392+A-as Antisense BS-469+A3-s Sense BS-469+A3-as Antisense

TGAGTATTGTTTTTTTTTTTTGTTAGT ACTCACCCTACCCAAATACC TATTAAYGTTTTTTTTTAGGGGG ACAACAAAAACAAAACTCCCTT TAAGGGAGGATTAGTTTTAGGG AACAAAAAACCTAACAAACCC TTTGAAGAAGGTTTTTATTTTTT CTTCCTCACAAATACCTAACC TTTAGTTTTGATTGTGGGGAGG ATTTTCCCAAAAACCCCCT TAATTAGGAAAAGAGATGGGG CACTAAAACCACTATCTAACCCA TGGGTAGTAGTGTTTTGTTTTG CACAATACCTTCAAAATACCAA TTTTAGGGTGATGGGAAAGG CTACCCACACCCAAATAAACAC

Primers for MSP (Methylation Specific PCR): TATCGAGTTAACGCGGGATAC MSP-160-ms Sense ACTTAAATCCCTCGACGCAT MSP-160-mas Antisense TTATATTGAGTTAATGTGGGATAT MSP-160-us Sense AAAACTTAAATCCCTCAACACAT MSP-160-uas Antisense AGGTTTTCGGTTGTTTTGTC MSP-283-ms Sense AAAAACTTCCGCTCGTTT MSP-283-mas Antisense GAAAGGTTTTTGGTTGTTTTG MSP-283-us Sense ACAAAAACTTCCACTCATTT MSP-283-uas Antisense GTTTTTTCGAAGGATCGC MSP-346-ms Sense ACGAATTACCCCGAATACTTT MSP-346-mas Antisense GTTTTTTGAAGGATTGT MSP-346-us Sense ACAAATTACCCAAATACTTT MSP-346-uas Antisense Primers for quantitative RT-PCR: U6-s Sense U6-as Antisense GAPDH-rt-s Sense GAPDH-rt-as Antisense Uc.160-rt-s Sense Uc.160-rt-as Antisense Uc.283-rt-s Sense Uc.283-rt-as Antisense Uc.346-rt-s Sense Uc.346-rt-as Antisense Primers for ChIP:

CTCGCTTCGGCAGCACA AACGCTTCACGAATTTGCGT TCTTCTTTTGCGTCGCCAG AGCCCCAGCCTTCTCCA GGCTTCAATGCACTATTGCA AAACCCTCCCTTGCTGACA AGCAAGTCTAATGGCGCATA CATAAAGGCTTTCCGCCA GGATTGCGTCGCTCTGA GCACTGTGAAGCCCATTGT

ChIP-160-s ChIP-160-as ChIP-283-s ChIP-283-as ChIP-346-s ChIP-346-as ChIP-160-1028bp-s ChIP-160-1028bp-as ChIP-160-114bp-s ChIP-160-114bp-as ChIP-160+226bp-s ChIP-160+226bp-as ChIP-283-1116bp-s ChIP-283-1116bp-as ChIP-283-118bp-s ChIP-283-118bp-as ChIP-283+363bp-s ChIP-283+363bp-as ChIP-346-1059bp-s ChIP-346-1059-as ChIP-346-0bp-s ChIP-346-0bp-as ChIP-346+271bp-s ChIP-346+271bp-as

Sense Antisense Sense Antisense Sense Antisense Sense Antisense Sense Antisense Sense Antisense Sense Antisense Sense Antisense Sense Antisense Sense Antisense Sense Antisense Sense Antisense

CCACAATGAGCACTGTTTCC CATTTCTCTCCACGCGATTT ACCTGCTAGCCGCTCCTC AACTTCCGCTCTTTCAGAG CGTACATTGTCCCACTCAGC AAGAGAAAGGCGGATTACCC AAAGGCGGGTCTCAGGAATA TGTCTGGCTTTGGACAGGAG

CCACAATGAGCACTGTTTCC CATTTCTCTCCACGCGATTT ATCCTCCTCCAGGACTACCC CACCTGAGATCAGGGGAGAA GGAGCTCGGCTGGTTATCTA CAATCCTCTCCCAGTCCAAA ACCTGCTAGCCGCTCCTC AACTTCCGCTCGCTTTCAGAG TCTTCTGATGCATCTTGAGTGG CCTGGCGGAAAGCCTTTAT

ATGGGGTGGTGAACAAAGTC TTCCTCACAGGTGCCTAACC GCGGAGGAACAGAGACAGAC TTCTCCTTCCTTGGCTGCTA GAAACAGACACCGGGAGACA GCCCCAATATGAGCGTAACA

Primers for MspI sensitivity assay: Nucleo-160-s Sense Nucleo-160-as Antisense Nucleo-283-s Sense Nucleo-283-as Antisense Nucleo-346-s Sense Nucleo-346-as Antisense

CCACAATGAGCACTGTTTCC GTGTGGGTGGAGTGGAGACT TGAGGGAGACAGGGAGAGAG AAACTTCCGCTCGTTTCAGA TCCGTACATTGTCCCACTCA GGTGCTGGAAGTGGAAGTGA

Primers for 5’ RACE: R-160-GSP1 Antisense R-160-NGSP1 Antisense R-283-GSP1 Antisense R-283-NGSP1 Antisense R-346-GSP1 Antisense R-346-NGSP1 Antisense

TTGGGCTAAGTAGGCAGGTACATGGCGG GCCGCAGGCGGTGGATGGATGAGATTTA ACTATTAAGTGCTTCCCTGGCGGAAAGC ACCCACTCAAGATGCATCAGAAGATGCA CACTGTGAAGCCCATTGTGGGCCTG GTTCCAGAGGCCTTCTCCAGCCTT

Primers for Uc.283+A 3’ primer walking: PW_Uc.283_1_1 Antisense PW_Uc.283_1_2 Antisense PW_Uc.283_1_3 Antisense PW_Uc.283_1_4 Antisense PW_Uc.283_1_5 Antisense PW_Uc.283_1_6 Antisense

GCGGTGGGACCCTTTGCAATTACTTTT CCACCACCATCACCCCCAAAAACATAC GTGACTCGGGAAGTGGCCAGATGTCTT ACCCCGCAGCGTAGTTTCTGGCTCTCT GGAAATAAAACATCGCCGGCTGTCAGA AATTAGAGGCGTGACACCAGCATCTGT

R_Uc.283_NGSP2

Sense

Primers for Uc.283+A 3’ cloning: C_Uc283_EcoRI_s Sense C_Uc283_NotI_a

Antisense

TGCATCTTCTGATGCATCTTGAGTGGGT

AAAAAAAAGAATTCGGAGCGGGACTGGT CTTCCTCCCGGCTGTTTTCGGAGGGG AAAAAAAAGCGGCCGCAATTAGAGGCGT GACACCAGCATCTGTGCTGGGAGGATTC

Supplementary Table 3

Location

Start

End

Strand

Upstream gene

Downstream gene

Uc.160+

chr5q14.1

77,304,600

77,304,921

+

AK128395 (196,673)

AP3B1 (28,984)

Uc.346+

chr12q23.3

105,500,640

105,500,841

+

RPC2 (72,637)

RFX4 (320)

Uc.283+A

chr10q11.23

50,274,763

50,275,039

-

AJ237663 (69,224)

ERCC6 (61,675)

T-UCR Name

Supplementary Table 3. Genomic context of the identified CpG island hypermethylated T-UCRs. The chromosomal location, the start and the end of the ultraconserved region of the transcript, the transcribed strand and the closest upstream and downstream protein-coding genes are represented (in parenthesis the distance between the probe and the transcription start site of the genes is shown).