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