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===Summary for using with T7tspn-top2: (USED)=== Sequences: 5'- [CATGAGA][TTAATACGACTCACTATAG][GGAGA][TCCTCCCTCGCGCCATCA][G][AGATGTGTATAAGAGACAG] -3β (T7tspn-top2) spcr T7 forward required spcr read sequence spcr ME 5'- [phos]CTGTCTCTTATACACATCT -3' (T7tspn-bot) T7 description: +1 CATGAGA[TTAATACGACTCACTATA|G|GGAGA] [end sequence for optimal binding] T7 --> [front sequence for optimal binding] After Annealing: 5β- CATGAGATTAATACGACTCACTATAGGGAGATCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG -3β (T7tspn-top2) 3'- TCTACACATATTCTCTGTC [Phos]-5' (T7tspn-bot) 5'- [phos] CTGTCTCTTATACACATCT -3' 3β- GACAGAGAATATGTGTAGAGACTACCGCGCTCCCTCCTAGAGGGATATCACTCAGCATAATTAGAGTAC -5β Fragments created after transposition: 5β- CATGAGA[TTAATACGACTCACTATAGGGAGA]TCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNN------ CTGTCTCTTATACACATCT -3' 3'- TCTACACATATTCTCTGTC ------NNNNNN GACAGAGAATATGTGTAGAGACTACCGCGCTCCCTCCT[AGAGGGATATCACTCAGCATAATT]AGAGTAC -5β Upon single insertion, T7 still present in either direction of insertion, IVT off top strand, and bottom: NNNNN------ CTGTCTCTTATACACATCT -3' 5β- CATGAGA[TTAATACGACTCACTATAGGGAGA]TCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNNN NNNNNNNNNNN GACAGAGAATATGTGTAGAGACTACCGCGCTCCCTCCT[AGAGGGATATCACTCAGCATAATT]AGAGTAC -5β 3'- TCTACACATATTCTCTGTC ------NNNN After fill in with taq polymerase (2X) (could use BST, T4 DNA poly also) 5β- CATGAGA[TTAATACGACTCACTATAGGGAGA]TCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNNNNN 3'- GTACTCT[AATTATGCTGAGTGATATCCCTCT]AGGAGGGAGCGCGGTAGTCTCTACACATATTCTCTGTC (NNNNNN)NNNN NNN(NNNNNN) CTGTCTCTTATACACATCTCTGATGGCGCGAGGGAGGA[TCTCCCTATAGTGAGTCGTATTAA]TCTCATG -3' NNNNNNNNNNN GACAGAGAATATGTGTAGAGACTACCGCGCTCCCTCCT[AGAGGGATATCACTCAGCATAATT]AGAGTAC -5β After IVT, and possibly RNA fragmentation. Addition of PolyA with E. coli PolyA polymerase (Ambion kit). Can also try adding adapter to 3'OH end with T4 RNA ligase. All RNA strands will have same 5' end. First G in GGG is the +1 site for the beginning of RNA synthesis for T7. RNA 5β- GGGAGATCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNNNNN... -3' RNA 5β- GGGAGATCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNNNNN AAAAAAAAAAAAAAAAAAAAAAAA -3' T7-frag-block-top2 CTGTCTCTTATACACATCTCTGATGGCGCGAGGGAGGATCTCCC/3InvdT/ POLY-A METHOD Adaptors for first/second strand cDNA synthesis, and addition of lawn adaptors (second strand synthesis) ILA adaptor blue (T7-top2-PCR-iaf)Tm=86.59 5β- [AATGATACGGCGACCACCGA][GATCT][CTCCCTCGCGCCATCAGAGAT] -3β(Tm=66.79 comp region only) X X XXXXX RNA 5β- GGG][AGATC][CTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNNNNN AAAAAAAAAAAAAAAAAAAAA -3' (polyA only Tm=49-52) (T20VN_PE_R)(Tm=78.92) 3'- NV TTTTTTTTTTTTTTTTTTTTTCTAGCC[TTCTCGCCAAGTCGTCCTTACG]-Bio-5' PCR_R.N2Ind[XX] (Tm=86.32) 3'- [TTCTCGCCAAGTCGTCCTTACG][GCTC][XXXXXX][TAGAGCATACGGCAGAAGACGAAC] -5' adaptor2 Tm=64.41 spr bc ILA adaptor orange Sequencing primers: T7tspn-read1 was designed off of template by mistake. however did not make a difference. (Nextera_R1) 5'- GCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG -3' XX (T7tspn-read1) 5'- TCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG -3' (N2IndSeq) XX 5'- AAGAGCGGTTCAGCAGGAATGCCGAG -3' 5β- AATGATACGGCGACCACCGAGATCTCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNNNNN AAAAAAAAAAAAAAAAAAAAAGATCGGAAGAGCGGTTCAGCAGGAATGCCGAG[XXXXXX]ATCTCGTATGCCGTCTTCTGCTTG -3' 3'- TTACTATGCCGCTGGTGGCTCTAGAGAGGGAGCGCGGTAGTCTCTACACATATTCTCTGTC NNNNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTCTAGCCTTCTCGCCAAGTCGTCCTTACGGCTC[XXXXXX]TAGAGCATACGGCAGAAGACGAAC -5' RANDOM NONAMER METHOD Adaptors for first/second strand cDNA synthesis, and addition of lawn adaptors (second strand synthesis) ILA adaptor blue (T7-top2-PCR-iaf)Tm=86.59 5β- [AATGATACGGCGACCACCGA][GATCT][CTCCCTCGCGCCATCAGAGAT] -3β(Tm=66.79 comp region only) X X XXXXX RNA 5β- GGG][AGATC][CTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN... -3' XXXXXX XXXXXXXXXXXX (N9_PE_R)(Tm=low) 3'-NNNNNNNNN CTAGCC[TTCTCGCCAAGTCGTCCTTACG] -5'-Bio PCR_R.N2Ind[XX] (Tm=86.32) 3'- [TTCTCGCCAAGTCGTCCTTACG][GCTC][GAACAT][TAGAGCATACGGCAGAAGACGAAC] -5' adaptor2 Tm=64.41 spr bc ILA adaptor orange RANDOM NONAMER METHOD Sequencing adaptors (second strand synthesis) T7tspn-Read1 (Tm=75.80) ILA adaptor blue TC][CTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG 5β- [AATGATACGGCGACCACCGA][GATCT][CTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNNNNNNNNNNNNNNNNNNN GATCGG[AAGAGCGGTTCAGCAGGAATGC][CGAG][CTTGTA][ATCTCGTATGCCGTCTTCTGCTTG] -5' [TTACTATGCCGCTGGTGGCT][CTAGA][GAGGGAGCGCGGTAGTCTCTACACATATTCTCTGTC NNNNNNNNNNNNNNNNNNNNNNNNNN CTAGCC[TTCTCGCCAAGTCGTCCTTACG][GCTC][GAACAT][TAGAGCATACGGCAGAAGACGAAC] adaptor2 Tm=64.41 spr bc ILA adaptor orange read primers from sequence reads etc
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