• Keine Ergebnisse gefunden

2. Materials

2.9 Oligonucleotides

Table 2.1 RNA-oligonucleotides. The siRNAs were obtained from Sigma-Aldrich.

siRNA oligonucleotides

human hnRNPL 3′ UTR 1581 GACAUUUCUCUUUCCUUUATT luciferase GL2 CGUACGCGGAAUACUUCGATT

Table 2.2 DNA-oligonucleotides. Desalted dry oligonucleotides were chemically synthesized (Sigma-Aldrich). All the oligonucleotides given represent the sequence from 5 to 3 end.

Real-time PCR primers Housekeeping genes

ACTB fwd GTGGAACGGTGAAGGTGACA

ACTB rev GGGACTTCCTGTAACAACGCA

GAPDH fwd TGCACCACCAACTGCTTAGC

GAPDH rev GGCATGGACTGTGGTCATGAG

snoRD U78 fwd GTGTAATGATGTTGATCAAATGT

snoRD U78 rev TTCTTCAGTGTTACCTTTGTCTA

U6 fwd CTCGCTTCGGCAGCACATA

U6 rev GCTTCACGAATTTGCGTGTCA

Plt-circR4

circ control fwd GGAGGTGACAACATGGAGGG

circ control rev CCGGCTGCCAACATAATCTGC

precursor control fwd GGAGGTGACAACATGGAGGG

precursor BGH rev AGGAAAGGACAGTGGGAGTG

Tornado

(CA)20 circ fwd TCGGCGTGGACTGTAGAAC

(CA)20 circ rev TGTGTGTGTGTGTGGCGG

(CA)100 Broccoli circ fwd GGTCGGCGTGGACTGTAG

(CA)100 Broccoli circ rev GATACGAATATCTGGACCCGACCGTC CA-SELEX X2 circ fwd TCGGCGTGGACTGTAGAAC

CA-SELEX X2 circ rev CATGTATGCGGCCGCACT CA-SELEX X4 circ fwd CAAGATGTGGCCGCGGTC CA-SELEX X4 circ rev ATGTATGGATCCGCGGCCG CA-SELEX X4 Broccoli circ fwd GGTCGGCGTGGACTGTAG

CA-SELEX X4 Broccoli circ rev GATACGAATATCTGGACCCGACCGTC control Broccoli circ fwd GGTCGGCGTGGACTGTAG

control Broccoli circ rev GATACGAATATCTGGACCCGACCGTC CD45

CD45 total (ex7) fwd TGCCTACCTTAATGCCTCTGA CD45 total (ex7) rev TGGTTGAAATGACAGCGCTT

30 RT-PCR primers

Housekeeping and other endogenous genes

ACTB fwd TGGACTTCGAGCAAGAGATG

ACTB rev GTGATCTCCTTCTGCATCCTG

CAMSAP1 circ fwd CCCTGATGATGGCCTACACT

CAMSAP1 circ rev TGTGCTCCTGCTCATACTGG

CAMSAP1 lin rev GGACCTTTTGATGAGCTGGA

CAMSAP1 circ fwd CCCTGATGATGGCCTACACT

IFN-1 fwd TGGGAGGATTCTGCATTACC

IFN-1 rev CAGCATCTGCTGGTTGAAGA

GAPDH fwd GAGTCAACGGATTTGTGCGT

GAPDH rev GATCTCGCTCCTGGAAGATG

PKR fwd AGCGAACAAGGAGTAAGGGA

PKR rev AGCAAGAATTAGCCCCAAAGC

snoRD U78 fwd GTGTAATGATGTTGATCAAATGT

snoRD U78 rev TTCTTCAGTGTTACCTTTGTCTA

U1 fwd GCGAGGCTTATCCATTGCAC

U1 rev GCAGTCCCCCACTACCACAA

U6 fwd CTCGCTTCGGCAGCACATA

U6 rev GCTTCACGAATTTGCGTGTCA

Plt-circR4

circ control fwd GGAGGTGACAACATGGAGGG

circ control rev CCGGCTGCCAACATAATCTGC

precursor control fwd GGAGGTGACAACATGGAGGG

precursor BGH rev AGGAAAGGACAGTGGGAGTG

tricRNA

tricRNA Broccoli divergent fwd GTCGAGTAGAGTGTGGGCTCGT tricRNA Broccoli divergent rev GATACGAATATCTGGACCCGACCGTC tricRNA Broccoli prec fwd CCTTCGATAGCTCAGTTGGT

tricRNA Broccoli prec divergent

rev GATACGAATATCTGGACCCGACCGTC

PIE

(CA)100 circ fwd CACACACACAAGATCCACTA (CA)100 circ rev TGTGTGTGTGTGGATCCTCT

CA-SELEX circ fwd CACACGCAAGATCTCGAGAA

CA-SELEX circ rev TGAACCCACACGACCGTTTA

control circ fwd AGGATTGAGGATCGTTTCGCA control circ rev CGGACTGGCTTTCTACGTGT Tornado

(CA)20 circ fwd TCGGCGTGGACTGTAGAAC

(CA)20 circ rev TGTGTGTGTGTGTGGCGG

(CA)100 Broccoli circ fwd GGTCGGCGTGGACTGTAG

31

(CA)100 Broccoli circ rev GATACGAATATCTGGACCCGACCGTC CA-SELEX X2 circ fwd TCGGCGTGGACTGTAGAAC

CA-SELEX X2 circ rev CATGTATGCGGCCGCACT CA-SELEX X4 circ fwd CAAGATGTGGCCGCGGTC CA-SELEX X4 circ rev ATGTATGGATCCGCGGCCG CA-SELEX X4 Broccoli circ fwd GGTCGGCGTGGACTGTAG

CA-SELEX X4 Broccoli circ rev GATACGAATATCTGGACCCGACCGTC control Broccoli circ fwd GGTCGGCGTGGACTGTAG

control Broccoli circ rev GATACGAATATCTGGACCCGACCGTC

control circ fwd AGGATGAGGATCGTTTCGCA

control circ rev CGGACTGGCTTTCTACGTGT

hnRNP L alternative splicing targets

ARGBP2 fwd CTTGGAGATCTTACAATGATGGC

ARGBP rev GTTTCCATTCACTGTTATCTGGC

BPTF (FALZ) fwd TCATCAAACCTTTGCTACATGG

BPTF (FALZ) rev CTGACTGGTACCTGTACTTGATG

CC2D2A fwd CCAGGGAAAGAGGTAGAAAGGAC

CC2D2A rev GTCCTCGGCATCATCACCAT

C5orf42 fwd CAGTTCCATCGTCTGCAGAGT

C5orf42 rev CTTGCAGCCGTTTCCACAAT

CARS2 fwd CTGCTGTGTTTGGTGCCATC

CARS2 rev TCCACCACACCATGCAAGG

DAF fwd TCCCACCAACAGTTCAGAAAC

DAF rev GTCCTGGAAACAGGTGTACTCC

HMMR fwd AGATACTACCTTGCCTGCTTCA

HMMR rev GCCTTGCTTCCATCTTTTCCA

DLG1 fwd GCATTGCTGGAGGTGTTGGA

DLG1 rev TGGTGATATCAGGTGGTGCA

GPBP1 fwd TCCTGAGTATGAGAGAGAACCAA

GPBP1 rev TCTTAACTGGCTGTGACGGA

PAPOLA fwd GAGTCAAGAGGACATCCTCACC

PAPOLA rev AGAGGCCAACAGAGAAGCC

PPP3CB fwd AGTGGAGTGTTAGCTGGAGGA

PPP3CB rev CCGAGGTGGCATTCTCTCAT

RIF1 fwd TCCATACCATGCCCAACAGA

RIF1 rev GAGTTGTCCCAGGCCTCTTG

TJP1 fwd ATATCCTCCTTACTCACCACAAG

TJP2 rev TTCAAAACATGGTTCTGCCTC

WHSC1 fwd TTCCACGGCAGCATCTTCAG

WHSC1 rev AGTCACTCCTCGCTCAGACT

CD45

CD45 ex2/3 fwd GGCAAAGCCCAACACCTTCCCCCACTG

CD45 ex7 rev GGGCTCAGAGTGGTTGTTTC

CD45 ex2/3 fwd (for circRNA Ex7) AGAAGTATTTGTGACAGGGCA CD45 ex7 rev (for circRNA Ex7) TGAAATGACAGCGCTTCCAG

CD45 ex5 rev GAAGGTGAGGCGTCTGTACT

32

CD45 ex5 fwd AGTACAGACGCCTCACCTTC

CD45 ex9 rev CTGAAACTTTTCAACCCCTGGTGGCAC

circRNA Ex4 ESS fw GCAGTGGTGTGAGTAGGCTT

circRNA Ex4 ESS rev CCCGCAAGCACCTTTGAG

circRNA Ex5 fw GTGGGAAGGTGAGGCTTACA

circRNA Ex5 rev CCTCTGCGCTTACTCCCTAC

Short-living mRNAs (actinomycin D-experiment)

FOXO3 fwd GACAAACGGCTCACTCTGTC

FOXO3 rev AGTTCCCTCATTCTGGACCC

MYC fwd GCTGCTTAGACGCTGGATTT

MYC rev CTCCTCCTCGTCGCAGTAG

ZNF260 fwd GCCATGGTCGTCTTTGTCTG

ZNF260 rev GGAACAAAGCAGCATCCCAA

Cloning primers Plt-circR4

5 repeats downstream fwd ATAGCGGCCGCAGGATGTGTTTGAGCCTAGTC 5 repeats downstream rev AGGAAAGGACAGTGGGAGTG

5 repeats upstream fwd AGTCTTAAGAGACCCAAGCTGGCTAGC 5 repeats upstream rev CCGGCTGCCAACATAATCTGC

Oligonucleotides for cloning Plt-circR4

2X upstream repeat fwd CTGGCTAGCAGGTTGGTGCAAAAGTAATTGCAGTTTTTAG GTTGGTGCAAAAGTAATTGCAGTTTTTGCTAGCCTG 2X upstream repeat rev CAGGCTAGCAAAAACTGCAATTACTTTTGCACCAACCTAA

AAACTGCAATTACTTTTGCACCAACCTGCTAGCCAG 2X downstream repeat fwd TAAGGGCCCAAAAATGCAATTACTTTTGCACCAACCTAAA

AATGCAATTACTTTTGCACCAACCTGGGCCCGTT

2X downstream repeat rev AACGGGCCCAGGTTGGTGCAAAAGTAATTGCATTTTTAGG TTGGTGCAAAAGTAATTGCATTTTTGGGCCCTTA

4X upstream repeat fwd

CTGGCTAGCCGCGGAGGTTGGTGCAAAAGTAATTGCAGT TTTTAGGTTGGTGCAAAAGTAATTGCAGTTTTTAGGTTGGT GCAAAAGTAATTGCAGTTTTTAGGTTGGTGCAAAAGTA 4X upstream repeats rev

CAGGCTAGCGGTACCAAAAACTGCAATTACTTTTGCACCA ACCTAAAAACTGCAATTACTTTTGCACCAACCTAAAAACTG CAATTACTTTTGCACCAACCTAAAAACTGCAATTAC

4X downstream repeats fwd

TAAGGGCCCCCTGCAGGAAAAATGCAATTACTTTTGCACC AACCTAAAAATGCAATTACTTTTGCACCAACCTAAAAATGC AATTACTTTTGCACCAACCTAAAAATGCAATTAC

4X downstream repeats rev

AACGGGCCCGCGGCCGCAGGTTGGTGCAAAAGTAATTGC ATTTTTAGGTTGGTGCAAAAGTAATTGCATTTTTAGGTTGG TGCAAAAGTAATTGCATTTTTAGGTTGGTGCAAAAG tricRNA

CA-SELEX51 SacII fwd GGATACATGACACACACACGCAGTGGCCGC CA-SELEX51 SacII rev GGCCACTGCGTGTGTGTGTCATGTATCCGC

33 PIE

CA-SELEX X4 in PIE fwd

TGAGGATCCATACATGACACACACACGCAATATATACATG ACACACACACGCAATATATACATGACACACACACGCAATA TATACATGACACACACACGCAAGATCTCGAGCTAA CA-SELEX X4 in PIE rev

TTAGCTCGAGATCTTGCGTGTGTGTGTCATGTATATATTG CGTGTGTGTGTCATGTATATATTGCGTGTGTGTGTCATGT ATATATTGCGTGTGTGTGTCATGTATGGATCCTCA

Tornado

CA-SELEX X4 fwd

GGCCGCGGATCCATACATGACACACACACGCAATATATA CATGACACACACACGCAATATATACATGACACACACACGC AATATATACATGACACACACACGCAAGATGTGGCCGC CA-SELEX X4 rev

GGCCACATCTTGCGTGTGTGTGTCATGTATATATTGCGT GTGTGTGTCATGTATATATTGCGTGTGTGTGTCATGTATA TATTGCGTGTGTGTGTCATGTATGGATCCGC

CA-SELEX X4 Broccoli fwd

ATGATGGGTCCCAGAGGATCCATACATGACACACACAC GCAATATATACATGACACACACACGCAATATATACATGAC ACACACACGCAATATATACATGACACACACACGCAAGAT CTCGAGAAGGGTCCCATCATT

CA-SELEX X4 Broccoli rev

AATGATGGGACCCTTCTCGAGATCTTGCGTGTGTGTGTC ATGTATATATTGCGTGTGTGTGTCATGTATATATTGCGTG TGTGTGTCATGTATATATTGCGTGTGTGTGTCATGTATG GATCCTCTGGGACCCATCAT

CA-SELEX X2 fwd GGCCGCATACATGACACACACACGCAATACATGACACAC ACACACGCAGTCGCCGC

CA-SELEX X2 rev GGCCACTGCGTGTGTGTGTCATGTATTGCGTGTGTGTGT CATGTATGC

(CA)20 fwd GGCCGCCACACACACACACACACACACACACACACACA

CACACACAGT

(CA)20 rev GGCCACTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGT

GTGTGTGGC

Oligonucleotide templates for preparation of antisense circRNA CD45

Ex4 3′SS 30 OR fwd TAATACGACTCACTATAGGGAGTAAGCTTGCTGTAGTCA ATCCTGTTAATTAATGGAAAAGCTTACAGTA

Ex4 3′SS 30 OR rev TACTGTAAGCTTTTCCATTAATTAACAGGATTGACTACAG CAAGCTTACTCCCTATAGTGAGTCGTATTA

Ex4 ESS 30 OR fwd TAATACGACTCACTATAGGGAGTAAGCGGTGCTTGCGGG TGAGAATGCAGTGGTGTGGCTTACAGTA

Ex4 ESS 30 OR rev TACTGTAAGCCACACCACTGCATTCTCACCCGCAAGCACC GCTTACTCCCTATAGTGAGTCGTATTA

Intr4 30 OR fwd TAATACGACTCACTATAGGGAGTAAGCCATCAGTCTTCCC CCACTGGAGATGAATAAGCTTACAGTA

Intr4 30 OR rev TACTGTAAGCTTATTCATCTCCAGTGGGGGAAGACTGATG GCTTACTCCCTATAGTGAGTCGTATTA

Scrambled 30 OR fwd TAATACGACTCACTATAGGGAGTAAGCGTGCGGCATGAA GTGGCGGTTGAGGTGTGTGCTTACAGTA

Scrambled 30 OR rev TACTGTAAGCACACACCTCAACCGCCACTTCATGCCGCAC GCTTACTCCCTATAGTGAGTCGTATTA

Ex4 3′SS 40 OR fwd TAATACGACTCACTATAGGGAGTAAGCCATCTTTGCTGTA GTCAATCCTGTTAATTAATGGAAAATGGCTTACAGTA

34

Ex4 3′SS 40 OR rev TACTGTAAGCCATTTTCCATTAATTAACAGGATTGACTACA GCAAAGATGGCTTACTCCCTATAGTGAGTCGTATTA Ex4 ESS 40 OR fwd TAATACGACTCACTATAGGGAGTAAGCTCAAAGGTGCTTG

CGGGTGAGAATGCAGTGGTGTGAGTAGGCTTACAGTA Ex4 ESS 40 OR rev TACTGTAAGCCTACTCACACCACTGCATTCTCACCCGCAA

GCACCTTTGAGCTTACTCCCTATAGTGAGTCGTATTA Ex4 5′SS 40 OR fwd TAATACGACTCACTATAGGGAGTAAGCAACAACTTTTGTG

TGCCAACCTGTGGTATTAAAAGCACTAGCTTACAGTA Ex4 5′SS 40 OR rev TACTGTAAGCTAGTGCTTTTAATACCACAGGTTGGCACAC

AAAAGTTGTTGCTTACTCCCTATAGTGAGTCGTATTA Intr4 40 OR fwd TAATACGACTCACTATAGGGAGTAAGCGAGATCATTACAT

CAGTCTTCCCCCACTGGAGATGAATAAGCTTACAGTA Intr4 40 OR rev TACTGTAAGCTTATTCATCTCCAGTGGGGGAAGACTGATG

TAATGATCTCGCTTACTCCCTATAGTGAGTCGTATTA Ex6 ESS 40 OR fwd TAATACGACTCACTATAGGGAGTAAGCAACTGGGTCTGTA

GGAAAGGTGCTGGCTGTACTCCTCTCTGCTTACAGTA Ex6 ESS 40 OR rev TACTGTAAGCAGAGAGGAGTACAGCCAGCACCTTTCCTAC

AGACCCAGTTGCTTACTCCCTATAGTGAGTCGTATTA Scrambled 40 OR fwd TAATACGACTCACTATAGGGAGTAAGCCGGTGAGGTGGA

TATTAACGGTGGCAAGGTCATGTGAGGTGCTTACAGTA Scrambled 40 OR rev TACTGTAAGCACCTCACATGACCTTGCCACCGTTAATATC

CACCTCACCGGCTTACTCCCTATAGTGAGTCGTATTA

Ex5 40 OR fwd TAATACGACTCACTATAGGGAGTAAGCGCAGAGGGCGTC

TGCGAGTCTGCGTGCGTGGGAAGGTGAGGCTTACAGTA

Ex5 40 OR rev TACTGTAAGCCTCACCTTCCCACGCACGCAGACTCGCAG

ACGCCCTCTGCGCTTACTCCCTATAGTGAGTCGTATTA

Ex7 40 OR fwd TAATACGACTCACTATAGGGAGTAAGCGGGCTCAGAGTG

GTTGTTTCAGAGGCATTAAGGTAGGCATGCTTACAGTA Ex7 40 OR rev TACTGTAAGCATGCCTACCTTAATGCCTCTGAAACAACCA

CTCTGAGCCCGCTTACTCCCTATAGTGAGTCGTATTA Oligonucleotides for circRNA qPCR standards (absolute quantification)

Tornado

T7 OR CA-SELEX X4 Broccoli fwd

TAATACGACTCACTATAGGGAGTAAGCAACCATGCCGAGT GCGGCCGCTTGCCCGGAGACGGTCGGGTCCAGATATTC GTATCTGTGGCCGCGGTCGGCGTGGACTGTAGGCTTACA GTA

T7 OR CA-SELEX X4 Broccoli rev

TACTGTAAGCCTACAGTCCACGCCGACCGCGGCCACAGA TACGAATATCTGGACCCGACCGTCTCCGGGCAAGCGGCC GCACTCGGCATGGTTGCTTACTCCCTATAGTGAGTCGTAT TA

T7 OR CA-SELEX X4 fwd

TAATACGACTCACTATAGGGAGTAAGCAACCATGCCGAG TGCGGCCGCGGATCCATACATGCAAGATGTGGCCGCGG TCGGCGTGGACTGTAGGCTTACAGTA

T7 OR CA-SELEX X4 rev

TACTGTAAGCCTACAGTCCACGCCGACCGCGGCCACATC TTGCATGTATGGATCCGCGGCCGCACTCGGCATGGTTGC TTACTCCCTATAGTGAGTCGTATTA

35 Oligonucleotides for Northern blot riboprobe tricRNA

Riboprobe CA-SELEX fwd

GGCTCGTGGCCGCGGATACATGACACACACACGCAGTG GCCGCGGTGTTGAAAGTGCGGCCGCGAGACGGTCGGGT CCAGATATTCGTATCTGTCGAGTACCCTATAGTGAGTCGT ATTA

Riboprobe CA-SELEX rev

TAATACGACTCACTATAGGGTACTCGACAGATACGAATAT CTGGACCCGACCGTCTCGCGGCCGCACTTTCAACACCGC GGCCACTGCGTGTGTGTGTCATGTATCCGCGGCCACGAG CC

*OR – stem sequence from Jost et al. (2018)

36