Oligonucleotides,Location,Sequence (5`->3`),Purpose,Melting Temp.,%GC,
ps1_piwi,piwi locus upstream of cloning region,GAAAGCCAGTTCACACGGTT,genotyping,63.6,50,
pas2_piwi,piwi locus downstream of cloning region,ATAATCAAAGATGAAGCAGAAAAAA,genotyping,59.6,24,
pas3_intron,inside of intron,AAGCATTTTTTTCACTGCATT,genotyping,59.2,28.6,
ps4_flag,"inside flag tag, sense",GACTACAAAGACCATGACGG,sequencing/ genotyping,60.4,50,
pas5_flag,"inside flag tag, antisense",CCGTCATGGTCTTTGTAGTC,RT-PCR,60.4,50,
ps6_UHA,inside upstream homology arm,CTGAGTCCAAAGCGTCGTTT,RT-PCR,63.2,50,
pas7_tag,"inside tag, antisense",AACCTCCAGATCCACCCTTG,RT-PCR,63.9,55,
ps8_pp0,"inside plasmid, upstream of intron, sense",GAGTCAGTGAGCGAGGAA,sequencing ,61,55.6,
ps9_pp1,"inside plasmid, upstream of intron, sense",TAATGTGAGTTAGCTCACTC,sequencing,57.3,40,
ps10_puro,"inside puromycin resistance gene, sense",GTTCTTGCAGCTCGGTGAC,sequencing,63.4,57.9,
ps11_prom,"inside of Act5C promoter, sense",ATGCTCTCCGCTCTCAAG,RT-PCR/ genotyping,61.5,55.6,
ps12_sgRNA,"Sense oligo for sgRNA cloning, lowercase lettes signify cloning overhangs",cttcGCGAGTGCCAAAAAGTAACAA ,,,,
pas13_sgRNA,"Antisense oligo for sgRNA cloning, lowercase lettes signify cloning overhangs",aaacTTGTTACTTTTTGGCACTCGC ,,,,
ps14_piwi-LHA,piwi left homology arm genomic PCR sense,tatgaccatgattacgccaagcttgcatgcTCAAAATATTCAATTTCCAA,,,,
pas15_piwi-LHA,piwi left homology arm genomic PCR antisense,atgtctggatcacttacctggTGTTACTTTTTGGCACTCGC,,,,
ps16_piwi-RHA,piwi right homology arm genomic PCR sense,gctctggctcttcaggctcaGCTGATGATCAGGGACGT,,,,
pas17_piwi-RHA,piwi right homology arm genomic PCR antisense,tgcaggcatgcagaagacatGTATGCTTTGCCTGCAATTTC,,,,
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Plasmids,Notes,,,,,
pPuro<3FH-BbSI,Empty donor plasmid. Contains the selection intron for Drosophila melanogaster and 3xFlag-3xHA (FH-) epitope tag. Both are flanked by BbsI restrictions sites to enable cloning of homology arms using HIFI system. The selectable marker (puromycin) is located in the intron and is driven by its own minimal Act5C promoter. ,,,,,
pPuro<3FH-piwi,Donor plasmid to endogenously tag piwi gene in Drosophila melanogaster ovarian somatic sheet cells (OSC). ,,,,,
p>sgRNApiwi,Plasmid expressing the sgRNA GCGAGTGCCAAAAAGTAACAA to edit piwi at the 5' end,,,,,
p>Cas9(w/oPuro),Plasmid expressing Cas9 without puromycin resistance. To be used with puromycin resistance containing donor plasmids.,,,,,
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Adaptors for Illumina sequencing,,,,,,
5’ hybrid adaptor,Used in all libraries,5’-GTTCAGAGTTCTACAGTCCGACGArUrCrNrNrNrNrNrNrNrN-3’,,,,
3’ Index adaptor 29.04 ,Used in eFH-Piwi library,5’-NNGCGATATGGAATTCTCGGGTGCCAAGG-3’,,,,
3’ Index adaptor 29.31 ,Used in eFH-Piwi library,5’- NNTAGCGATGGAATTCTCGGGTGCCAAGG-3’,,,,
3’ Index adaptor 29.32 ,Used in eFH-Piwi library,5’- NNCTGTAGTGGAATTCTCGGGTGCCAAGG-3’,,,,
3’ Index adaptor 29.33 ,Used in WT Piwi library,5’- NNTAGTCGTGGAATTCTCGGGTGCCAAGG-3’,,,,
3’ Index adaptor 29.34 ,Used in WT Piwi library,5’- NNAGTGTCTGGAATTCTCGGGTGCCAAGG-3’,,,,
3’ Index adaptor 29.35 ,Used in WT Piwi library,5’- NNATCGACTGGAATTCTCGGGTGCCAAGG-3’,,,,
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