2. Material and methods
2.1. Material
2.1.6. Oligonucleotides
Unmodified DNA oligonucleotides used in this study were purchased from eurofins Genomics. Lyophilised DNA oligonucleotides were diluted in water at a stock concentration of 100 pmol/µl and stored at −20 °C.
Plasmid name Description Source/Reference
pRmHa-3-FLAG-HA-dCoREST-L
Encodes full length dCoREST-L, tagged N-terminally with a FLAG/HA-tag for inducible expression in Drosophila cells, driven by metallothionein promoter.
Corina Webert AG Brehm
pRmHa-3-FLAG-HA-dCoREST-M
Encodes full length dCoREST-M, tagged N-terminally with a FLAG/HA-tag for inducible expression in Drosophila cells, driven by metallothionein promoter.
Corina Webert AG Brehm
pRmHa-3-FLAG-HA-dLSD1
Encodes full length dLSD1, tagged N-terminally with a FLAG/HA-tag for inducible expression in Drosophila cells, driven by metallothionein promoter.
Corina Webert AG Brehm
pNLS-eGFP-C1 A pEGFP-C1 (Clontech) derived vector
that encodes eGFP. H. Leonhardt
pOT2-dCoREST-L cDNA encoding for full length
dCoREST-L in the pOT2 vector. IP20671
pRmHa-3-FLAG-HA-dL(3)mbt
Encodes full length dL(3)mbt, tagged N-terminally with a FLAG/HA-tag for inducible expression in Drosophila cells, driven by metallothionein promoter.
Corina Webert AG Brehm
pRmHa-3-FLAG-HA-dLint-1
Encodes full length dLint-1, tagged N-terminally with a FLAG/HA-tag for inducible expression in Drosophila cells, driven by metallothionein promoter.
Corina Webert AG Brehm
pRB17
Vector carrying the sequence of U6-promoter fused to T7 U6-promoter for CRISPR/Cas9 U6-sgRNA template generation.
Addgene #52527
pSK23
Vector carrying the sequence of GFP-tagged generation of homologous recombination template for tagging in CRISPR/Cas9 S2 cells.
Addgene #72851
pSK25
Vector carrying the sequence of 2×FLAG-tagged generation of homologous
recombination template for tagging in CRISPR/Cas9 S2 cells.
Addgene #72853
2.1.6.1. Primers used for CRISPR/Cas9 tagging
Oligonucleotides used in CRISPR/Cas9 tagging experiments were designed according to the protocol described previously (Böttcher et al., 2014).
Table 2.5. List of primers used in CRISPR/Cas9 tagging experiments.
Primer name Sequence Reference
sgRNA scaffold
GTTTAAGAGCTATGCTGGAAACAGCATAGCA AGTTTAAATAAGGCTAGTCCGTTATCAACTT GAAAAAGTGGCACCGAGTCGGTGC
(Böttcher et al. 2014) U6-promoter sense GCTCACCTGTGATTGCTCCTAC (Böttcher et al. 2014) sgRNA antisense gcttattctcAAAAAAGCACCGACTCGGTGC
CACT (Böttcher et al. 2014)
CRISPR act5c cctattttcaatttaacgtcgACCGCAAGTG
CTTCTAAGAgtttaagagctatgctg (Böttcher et al. 2014) act5c-sense TGGATCTCCAAGCAGGAGTACGACGAGTCCG
GCCCCTCCATTGTGCACCGCAAGTGCTTCgg atcttccggatggctcgag
(Böttcher et al. 2014)
act5c-antisense
CCTCCAGCAGAATCAAGACCATCCCGATCCT GATCCTCTTGCCCAGACAAGCGATCCTTCga agttcctattctctagaaagtataggaactt cCATATG
(Böttcher et al. 2014)
CRISPR dCoREST cctattttcaatttaacgtcgCAGAGTTCCT
GGCCAACTGgtttaagagctatgctg This study dCoREST-sense GCGAAGAAAATCGCGCTCAGCACCGGAGGCG
GAAGCAGCGTCGCAGAGTTCCTGGCCAACgg atcttccggatggctcgag
This study
dCoREST-antisense
ATGTTATGTATCGGTATATATCTATGCGTGC ATATATATCGCGAGTGAACACGTCGCTCCga agttcctattctctagaaagtataggaactt cCATATG
This study
CRISPR dLSD1 cctattttcaatttaacgtcgAACAATGTAT
TTAGCGTGAgtttaagagctatgctg This study dLSD1-sense TCGTCAAAGAAGTCGGAGGAGAATTCAAACT
CAAACACTGCCGACTCTACGGAGCTACAGgg atcttccggatggctcgag
This study
dLSD1-antisense
CAAAACTAAACGCTCTAGGAGTAACTGCTGG GGACCAAATGCATCACGCTAAATACATTGga agttcctattctctagaaagtataggaactt cCATATG
This study
CRISPR dL(3)mbt cctattttcaatttaacgtcgCCCTTGCGCA
CGTCCTCTTgtttaagagctatgctg This study dL(3)mbt-sense TCCGACGGCGATGTGGCGATGGTGCCGATGG
AAGTGCGCACGCCCTTGCGCACGTCCTCTgg atcttccggatggctcgag
This study
In lowercase letters the sequence that anneals to the template plasmids are shown, and in uppercase letters is the gene-specific DNA sequence.
Table 2.5. List of primers used in CRISPR/Cas9 tagging experiments. (continuation)
2.1.6.2. Primers for genotyping of tagged cell lines
To check the integration of tagging constructs in S2[Cas9] cells, gDNA isolated from cell lines was genotyped according to the protocol described previously (Böttcher et al., 2014).
Table 2.6. List of primers used for genotyping of S2[Cas9] cell lines.
Primer name Sequence Reference
dL(3)mbt-antisense
GGTGCAACAAAATAATCTTATAAATCAATCA ACGGAAGCGGATGCCTGGTATCCGGAGTCga agttcctattctctagaaagtataggaactt cCATATG
This study CRISPR dG9a cctattttcaatttaacgtcgGAGAAAATTG
GACACGCGTgtttaagagctatgctg This study CRISPR FLAG-dG9a cctattttcaatttaacgtcgGACAATTAAG
GCAAGATGAgtttaagagctatgctg This study dG9a-sense-FS CACCGGAAAATGAAACGGGAACGCTGTCGTC
TACAAATACGGAGAAAATTGGACACGCGTgg atcttccggatggctcgag
This study
dG9a-sense GCACCGGAAAATGAAACGGGAACGCTGTCGT
CTACAAATACGGAGAAAATTGGACACGCGgg atcttccggatggctcgag
This study
dG9a-antisense
TTTTATTTGTTGGATGAGACTGTGAAATCTG CAATCATCTCAGGTTTAGGTGGTTTTAGCga agttcctattctctagaaagtataggaactt cCATATG
This study
dG9a-antisense-NR
TTTTATTTGTTGGATGAGACTGTGAAATCTG CAATCATCTCAGGTTTAGGTGGTTTTAGCTC ATTTATCATCATCATCTTTATAATCggctcc ggaTTTA
This study
FLAG-dG9a-sense
ATCGAAAATCAAAGAAACTAATACGCAAAGT AATCAAATAGTGACAATTAAGGCAAGATGGA TTATAAAGATGATGATGATAAAtccggagcc GATTATA
This study
FLAG-dG9a-antisense
AGTAGCACAGTCGCTATTGAATGTACTGGAC ATGCTGTTCATCAGCTCAACAAAGTCTGTTT TATCATCATCATCTTTATAATCggctccgga TTTATCA
This study
In lowercase letters the sequence that anneals to the template plasmids are shown, and in uppercase letters is the gene-specific DNA sequence.
Primer name Sequence Reference
dCoREST-GFP_fw TCTCTTCTCCTCCTCCACAG
This study dCoREST-GFP_rv CGTCCCCCAAAACATCAATC
dLSD1-GFP_fw CCCAATCTATCTGACTCCTC
This study
dLSD1-GFP_rv TTACAGCGGCCTAGCTTCGT
dL(3)mbt-GFP_fw ATGGGGATGGCGATTGTGAA
This study dL(3)mbt-GFP_rv ATAATACCCGAATGGGCCGA
Table 2.6. List of primers used for genotyping of S2[Cas9] cell lines. (continuation)
2.1.6.3. Primers for generation of dsRNA by in vitro transcription (ivT) To knockdown specific proteins in Drosophila S2[Cas9] and S2 cells, dsRNA was used in RNAi experiments. Primers used for generation of gene-specific dsRNA by in vitro transcription had a T7 promoter sequence at the 5’ end.
Table 2.7. List of primers used for amplification of the templates for dsRNA synthesis.
Primer name Sequence Reference
dG9a-C-fw CACCGGAAAATGAAACGGGA
This study
dG9a-C-rv ACCGGGCTTCGATAACGATT
N-dG9a-fw CGATGCACAAATCTTGTCGG
This study
N-dG9a-rv TTAGCAAAGTACCACCCTCC
Primer name Sequence Reference
lig4-RNAi-fw taatacgactcactatagggCCCAATGATCCA AAGTGTTTTTGCA
(Böttcher et al. 2014) lig4-RNAi-rv taatacgactcactatagGGAAGTAGGATGCC
TTCGCGA
mus308-RNAi-fw taatacgactcactataggGCTGGGACTCCAC CGGAAAG
(Böttcher et al. 2014) mus308-RNAi-rw taatacgactcactatagggTACCGTCGCCGT
CCAGTAATG
EFGP-RNAi-fw gaattaatacgactcactatagggAGAGCTGG ACGGCGACGTAA
(Stielow at al. 2008) EFGP-RNAi-rv gaattaatacgactcactatagggAGACTTGT
ACAGCTCGTCCATG
dCoREST-RNAi-fw taatacgactcactatagggCATTCGCTCAGT TTTCTGACG
(Meier et al. 2012) dCoREST-RNAi-rv taatacgactcactatagggCCACCGAAATGT
ACTCCTCC
dCoREST-L-RNAi-fw taatacgactcactatagggAAGATTTGCAAC
GTGGTCTG Corina Webert
AG Brehm dCoREST-L-RNAi-rv taatacgactcactatagggTTCCGCCAAATA
GAGACTGG
dLSD1-RNAi-fw taatacgactcactatagggAAAGAAACGTCA ATCACCCG
(Meier et al. 2012) dLSD1-RNAi-rv taatacgactcactatagggCCTCTTCGTTGG
GTGTCATT
dL(3)mbt-RNAi-fw taatacgactcactatagggGTTGGTTTGGGT GCTGTCTT
(Meier et al. 2012) dL(3)mbt-RNAi-rv taatacgactcactatagggGCGTCTAAAGTT
CAGCCAGG
In lowercase letters the sequence of the T7 promoter is shown, and in uppercase letters the gene-specific DNA sequence in shown.
Table 2.7. List of primers used for amplification of the templates for dsRNA synthesis. (continuation)
2.1.6.4. Primers for gene expression analysis by qPCR
Oligonucleotides that were used in this study for qPCR experiments were designed at exon-intron borders for specific isoforms and their specificity was checked first using the primer BLAST tool (https://www.ncbi.nlm.nih.gov/tools/primer-blast) and then by control qPCR and agarose gel analysis.
Table 2.8. List of primers used for qPCR gene expression analysis.
Primer name Sequence Reference
dLint-1-RNAi-fw taatacgactcactatagggATGAAAGGGTCG
CTGGATT (Meier et al. 2012)
dLint-1-RNAi-rv taatacgactcactatagggGCTCGGCACTGG AATCAT
dG9a-RNAi-fw taatacgactcactatagggAAACCAAGTGTT ACTTTGAGAG
(Meier et al. 2012) dG9a-RNAi-rv taatacgactcactatagggTGTACAAAATAT
GCCACATCCT
In lowercase letters the sequence of the T7 promoter is shown, and in uppercase letters the gene-specific DNA sequence in shown.
Primer name Sequence Reference
Rp49-RT-fw TGTCCTTCCAGCTTCAAGATGACCATC
(Gabler et al., 2005) Rp49-RT-rv CTTGGGCTTGCGCCATTTGTG
dCoREST-RT-fw TCAAGGATGGCTCCGAGAAC
This study dCoREST-L-RT-rv TGTGCCATGCCCTTTCTTGT
dCoREST-RT-fw TCAAGGATGGCTCCGAGAAC
This study dCoREST-M-RT-rv CCTATTCTTCTGTATCTTGT
dLSD1-RT-fw ACGGCGAGTAGAGGAGAAAT
This study dLSD1-RT-rv GATTATGATGTCATCCGTCA
dL(3)mbt-RT-fw TTTCTGGCACCACATTTCTG
(Meier et al., 2012) dL(3)mbt-RT-rv CTCTCCTTCTGCGTACTCTGC
dLint-1-RT1-fw GCAGGAGCAGCAAAGACG
(Meier et al., 2012) dLint-1-RT1-rv CTCAAAGAGGCCGAGGAAC
dLint-1-RT2-fw CCGTGAAGCTGAAGGAGAAC
This study dLint-1-RT2-rv GGAAGTGCTTGCGAATAAGC
dG9a-RT-fw AACGATGACTTGGAGCGTGTA
Dr Karin Meier dG9a-RT-rv GGGAGTCAGCACGTTGAAGT