Supplementary information Supplementary Table S1 ...

Supplementary information

Locus PHKA1P1 EIF3FP3

forward primer 5'→3'

reverse 5'→3'

Restriction enzyme

GGGAGGATGTAGGAGTTATA ATTGTGAGGTTTATTTGTTATATT

ACCAAAATACTATACACATTATCTC CACTTCATCTTCTAACTCATTATAC

EcoRI HpaI

AGAGGTTAAGATGGAATTAAATATGTAAGG GTGAGGAGATTGAAGTATGAGATTAAGAAA GTTATTATTATTTGGAGATTTATTTATGTT

AAATAATCCAAACCCACTACCAAA CTACTTTACCACCTCACCAACTTC ACTTATCACAAAACCAAAAACC

HpyCH4III AlwⅠ MboⅠ

TGTTTGTATAAAATAGAAGGTATGG GAATATTTTGGATAATAGTGATTTT

AAAACTAAATTCCTACTCCACCTC CTACAATAAACAACATCTCCCAAC

EciI SnaBI

C1orf106- r TMEM183A- r

AAAGATTTGAAAGTGTATTGTGTAG AAGTGTTTGTGTAAATATGTGATTTAAAGT

CACACACTATAAACACCTATACCC TCATAAAACTCACCAACCCCAAAA

MboⅠ AclI

ZNF322P1 RALGAPA1P RPS28P7 UHRF1-r RPL18AP3

TTTAGTTAGTTTGAAAGGGTTTG GAGAGAGTTGTGTTGTTGTTTT TTTGGTTAGTTGGGTTAGATATTT TTAATGTGATATTTGTGTAAATATTT ATTAATTAATAATTTTATAAAGTTTAAAGAG

TCACAACACTCAATTCTTACCTCT CACCTTCTAAATAAACTTCTTCAC AATTATAACAAACCATTCCCATC CTTCTTCTTACTCTCTCTCAACC ACTAAAATACAAAATACCAAAAAC

BstBI Mbol AciI MaeIII MaeⅢ

TGGTAATAGTGATTTATTATGTTTTAAA TTTTAGGGTTTAATATTTAGTTTT

TCCTCATAAACTCCAACTACAAAA AACCTACTTCAACACCTTATACAC

EcoRⅠ TaqI

BMPR1APS2 AK4P3 Novel-r NUS1P1 GCSHP5

RBM8B H2BFS

Supplementary Table S1 PCR primer pairs and restriction enzyme used for COBRA

Annealing Locus

forward primer 5'→3'

reverse 5'→3'

PHKA1P1

AAGGGGGCAGGAGCGGCGA

TGAGAAATAGCCCGGGTGCCACCA

70

EIF3FP3

GTGAGGTTCATTTGTTACACTCGTA

GTTGGCGCTGGAACTGAG

60

TCCGAATGTTGGATGTACTGGAA

GAGGGCCCCAGAATCCGTTA

60

CACAGCGTTGAGAATTAAGAAGAT

GACAGGACAAACTATCATAAACTAG

64 67.5

*EIF3FP3(qPCR) BMPR1APS2

temp. (℃)

AK4P3

TAACTTTTCATACTGGCGAGAAGAAAAGAG

AGGAGTGAAGTATGGAAGCAGGC

Novel-r

GCTGAGTCAGCGCCACTG

CCATCCCGCCGTCCCGT

NUS1P1

GATGCTTGTGGCAGGAATTAT

GTACATCTGTAGCCTATGATTTGAGTCTA

C1orf106-r

GGACGAGTGAAGCAGAGTGG

CTGTCCACTAACCACAAGCATGGAT

72

TMEM183A-r

GGATTACGCCGACTCGGATCT

ATCGGATCACACAAGCCCGGAGACAGT

71

GGGGCAGCCTCGAGTGTAGT

60

*TMEM183A-r(qPCR) GGGCCACCTTATCTTAGTTCCAGA ZNF322P1

72 68.5

TTGTTCTGGGAGAAGCTAGGGC

TACTGCTGAAAGAGATGATAAGTGAAGT

67

UHRF1-r

GGAGCGATTTGTCTGGTTAATTCCG

ATGGACACCCGACTCGCTGA

71

*UHRF1-r(qPCR)

TAAGTTGTTCTCACTCTTCCCTCAG

AAAAGAGGACAAATTGATCTGGC

60

RBM8B

GTGAATTGATGGAGGGGCATAGG

CATTGTCCTCAGTGTGTCCAGC

70

H2BFS

GTCTGTGACATGGCTGGAGA

GAGACGAGCAGCAGATCGT

55

GGAGCGATTTGTCTGGTTAATTCCG

GAGCCCCAGCCTTCTCCATG

60

GAPDH(qPCR)

* Primer sets redesigned for quantitative PCR

Supplementary Table S2 PCR primer pairs used for RT-PCR and quantitative RT-PCR

template Locus EIF3FP3

BMPR1APS2

AK4P3

NUS1P1

TMEM183A-r

UHRF1-r

H2BFS

Primers

Usage

primer sequence 5'→3'

F

GGCGGTCCCAGCCTCT

3' RACE

R

GGGACACCCATTAAAGTGCTGACA

5' RACE

F

CACAGCGTTGAGAATTAAGAAGAT

5' RACE

nestF

TGATGGTTAAACCATCGGAGGAGAAACTC

5' RACE (nested PCR)

F

GGTCTGAATCGTCAGTGCATTATATTGACC 3' RACE

nestF

GGAGAGGTACGGGAAGTGGCCTGATG

3' RACE (nested PCR)

R

AGGAGTGAAGTATGGAAGCAGGC

5' RACE

nestR

GGTCAATATAATGCACTGACGATTCAGACC 5' RACE (nested PCR)

F

GTTTTGTGTGAAGCTACAGCATATCTAAT

5' RACE

nestF

TATATTATATTGAGGCATTTGTAGTGCC

5' RACE (nested PCR)

R

TTGTGATGCTTGTGGCAGGAATTAT

3' RACE

nestR

GTTTGCCTCTTCCTAGAACTTCAAT

3' RACE (nested PCR)

F

GGATTACGCCGACTCGGATCT

5' RACE

R

ATCGGATCACACAAGCCCGGAGACAGT

3' RACE

F

GGCCAGGCGTGTGG

3' RACE

nestF

ACGGACGGACGCCAGCACAT

3' RACE (nested PCR)

R

ATGGACACCCGACTCGCTGA

5' RACE

nestR

TGTTCCCAGCGAGGACGAG

5' RACE (nested PCR)

RT-F1

CAATGTGACATTTGTGCAAATACTC

confirmation of conection*

RT-R1

GCACTTGCACGTGACTTCA

confirmation of conection*

F

TGTCACAGACTTTTGTGGCAGCCCA

3' RACE

nestF

GCCTCAAGCCTTGGGAGCTT

3' RACE (nested PCR)

HSF2BPF GTCTGTGACATGGCTGGAGA

fusion check**

RT-R1

fusion check**

GAGACGAGCAGCAGATCGT

mRNA PC3

Brain

Placenta

Brain

Brain

Placenta

Brain

Testis

*confirmation of the conection between 3' and 5' RACE fragment **RT-PCR was carried out to check fusion transcript using primer for HSF2BP exon 6 and H2BFS.

Supplementary Table S3 PCR primer sequence to identify the structures of retrocopy associated transcripts

99964481

ZNF322P1

20709315

UHRF1-r

91361574

PHKA1P1

Novel-r

Novel-r

90993802

TSD

58480319

EIF3FP3

NUS1P1

chimp

TSD

human TSD

90045604-90049082

human

90990738

59434469-59438326

chimp 58476566

TSD

82402978

TSD

Chr. 21

human

44983061

TSD

Chr. 2

TSD

Chr. 14

RPL18AP3

EIF3FP3

chimp

91650884-91652881

human TSD

104659882

TSD

Chr. 1

91354714

human

RPS28P7

H2BFS

chimp 104657048

human

82399073

TSD

Chr. 12

TSD

PHKA1P1

108291998

TSD

44988086

H2BFS

TSD

AK4P3

chimp

TSD

Chr. 11

TSD

RPL18AP3

chimp

human

20703329

human RALGAPA1P

TSD

RPS28P7

chimp

TSD

104574989-104576435

Chr. 12

20921974-20924917

chimp

108280022

TSD

UHRF1-r

Chr. 9

104410387--104414373

chimp

human

TSD

chimp

RALGAPA1P

149703166

TMEM183A-r

TSD

2A: 59109711-59112015

chimp

95976492:95980593:1

Chr. 9

human

149697451

TSD

80601884-80605067

TSD

111906671

C1orf106-r

Chr. 14

29561532-29583420

TSD

ZNF322P1

99955562

human

111900295

168058487

TMEM183A-r

Chr. 12

human

31611275

TSD

31617393

AK4P3

TSD

Chr. X

48012905-48014983

TSD

GCSHP5

Chr. 3

56816390-56820003

chimp

human

168054773

chimp

C1orf106-r 115503829-115507544

Chr. 1

146430201-146432753

chimp

GCSHP5

47368157

TSD

human NUS1P1

47373982

TSD

Supplementary Figure S1 Dot plot analyses between human (x-axis) and chimp (y-axis) indicate human-specific retrocopy insertions. The chromosome positions are based on GRCh37 and CHIMP2.1.4. The arrowheads indicate the target site duplication (TSD) sequences flanking the insertion sites.