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===Sequences information, Using T7-nXTv2_i5-1-top and T7-nXTv2_i5-2-top=== Sequences: '''ORIGINAL T7tspn-top2: (69 bp)''' 5'- [CATGAGA][TTAATACGACTCACTATAG][GGAGA][TCCTCCCTCGCGCCATCA][G][AGATGTGTATAAGAGACAG] -3β (T7tspn-top2) spcr T7 forward required spcr read sequence spcr ME '''T7R1-NEXTERA XT V2 TOP (64 bp)''' 5'- [CATGG][AATTAATACGACTCACTATAGGGAGA]TCGTCGGCAGCGTC[AGATGTGTATAAGAGACAG] -3β (T7-nXTv2_i5-1-top) \---/ \-/\---------------------/ \--------------------------------/ spcr T7 rec. T7 req. forward read seq. \-----------------/ ME '''T7R1-NEXTERA XT V2 TOP + EXTRA (69 bp)''' 5'- [CATGG][AATTAATACGACTCACTATAGGGAGA][CTCCT][TCGTCGGCAGCGTC[AGATGTGTATAAGAGACAG] -3β (T7-nXTv2_i5-2-top) \---/ \-/\---------------------/ \---/ \--------------------------------/ spcr T7 rec. T7 req. forward spcr read seq. \-----------------/ ME '''NEXTERA XT V2 BOTTOM (19 bp with /5Phos/)''' 5'- /5Phos/CTGTCTCTTATACACATCT -3' (P-T7tspn-bot) '''T7 description:''' +1 [CATGG][AAT][TAATACGACTCACTATA|G|GGAGA] [end sequence] rec. T7 req --> [front sequence for optimal binding] '''After Annealing:''' 5'- CATGGAATTAATACGACTCACTATAGGGAGATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG -3β (T7-nXTv2-1-top) 3'- TCTACACATATTCTCTGTC/5Phos/-5' (P-T7tspn-bot) 5'- /5Phos/CTGTCTCTTATACACATCT -3' 3β- GACAGAGAATATGTGTAGACTGCGACGGCTGCTAGAGGGATATCACTCAGCATAATTAAGGTAC -5β '''Fragments created after transposition:''' 5'- CATGGAATTAATACGACTCACTATAGGGAGATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG NNNNNNNNNN--------- 5P/CTGTCTCTTATACACATCT -3' 3'- TCTACACATATTCTCTGTC/5P ---------NNNNNNNNNN GACAGAGAATATGTGTAGACTGCGACGGCTGCTAGAGGGATATCACTCAGCATAATTAAGGTAC -5β '''Upon single insertion, T7 still present in either direction of insertion, IVT off top strand, and bottom:''' 5'- NNN--------- 5P/CTGTCTCTTATACACATCT -3' 5'- CATGGAATTAATACGACTCACTATAGGGAGATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG NNNNNNNNNNNN -3' 3'- NNNNNNNNNNNN GACAGAGAATATGTGTAGACTGCGACGGCTGCTAGAGGGATATCACTCAGCATAATTAAGGTAC -5β 3'- TCTACACATATTCTCTGTC/5P ---------NNN -5' '''After fill in with taq polymerase (2X) (could use BST, T4 DNA poly also)''' 5'- CATGG[AATTAATACGACTCACTATAGGGAGA]TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG NNNNNNNNNNNNNN -3' 3'- GTACC[TTAATTATGCTGAGTGATATCCCTCT]AGCAGCCGTCGCAGTCTACACATATTCTCTGTC (NNNNNNNNN)NNN -5' 5'- NNN(NNNNNNNNN) CTGTCTCTTATACACATCTGACGCTGCCGACGA[TCTCCCTATAGTGAGTCGTATTAATT]CCATG -3' 3'- NNNNNNNNNNNNNN GACAGAGAATATGTGTAGACTGCGACGGCTGCT[AGAGGGATATCACTCAGCATAATTAA]GGTAC -5β '''After IVT, All RNA strands will have same 5' end. RNA is also purified after IVT.''' '''First G in GGG is the +1 site for the beginning of RNA synthesis for T7.''' '''for Tm's used used https://www.neb.com/tools-and-resources/interactive-tools/tm-calculator (NEB Tm calculator)''' '''All Tm's calculated using, "taq DNA polymerase", "taq 2X Mastermix"''' RNA 5β- GGGAGATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG NNNNNNNNNNNNN -3' '''After single strand cDNA synthesis to form cDNA/RNA hybrid''' RNA 5β- GGGAGATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG NNNNNNNNNNNN... -3' cDNA 3'- CCCTCTAGCAGCCGTCGCAGTCTACACATATTCTCTGTC NNNNNNNNNNNN... -5' '''After Rnase H nicking, denatuation of small RNAs? and addition of 5' end primer.''' '''Extension with BST or klenow exo- or DNAP1 or Taq2x (been using taq2x)''' DNA 5β- /5Phos/GGGAGATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG -3' (Tm=70, anneal@64C) (P-sss_nXTv2_prmr) cDNA 3'- CCCTCTAGCAGCCGTCGCAGTCTACACATATTCTCTGTC NNNNNNNNNNNN... -5' '''Now have double stranded DNA, which should be long.''' DNA 5β- /5Phos/GGGAGATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG NNNNNNNNNNNN... -3' cDNA 3'- CCCTCTAGCAGCCGTCGCAGTCTACACATATTCTCTGTC NNNNNNNNNNNN... -5' '''Custom Nextera i7 transposome''' i7 adapter 5β- GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG -3' (nXTv2_i7_top) 3'- TCTACACATATTCTCTGTC/5Phos/ -5' (P-nXTv2_i7_bot) 5'- /5Phos/CTGTCTCTTATACACATCT -3' (P-nXTv2_i7_bot) 3β- GACAGAGAATATGTGTAGAGGCTCGGGTGCTCTG -5' (nXTv2_i7_top) '''After custom nextera tagmentation:''' DNA 5β- /5Phos/GGGAGATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG NNN--------- 5P/CTGTCTCTTATACACATCT -3' cDNA 3'- CCCTCTAGCAGCCGTCGCAGTCTACACATATTCTCTGTC NNNNNNNNNNNN GACAGAGAATATGTGTAGAGGCTCGGGTGCTCTG -5' '''After fill in:''' DNA 5β- /5Phos/GGGAGATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG NNNNNNNNNNNN CTGTCTCTTATACACATCTCCGAGCCCACGAGAC -3' cDNA 3'- CCCTCTAGCAGCCGTCGCAGTCTACACATATTCTCTGTC NNNNNNNNNNNN GACAGAGAATATGTGTAGAGGCTCGGGTGCTCTG -5' '''Amplification (to add i5/i7 barcodes):''' '''for Tm's used used https://www.neb.com/tools-and-resources/interactive-tools/tm-calculator (NEB Tm calculator)''' '''All Tm's calculated using, "taq DNA polymerase", "taq 2X Mastermix"''' 5'- AATGATACGGCGACCACCGA -3' (i5_PCR_ILAdptr, Tm=59C) 5'- AATGATACGGCGACCACCGAGATCTACAC[i5]GGGAGATCGTCGGCAGC -3' (i5XX_T7-1_shrt, comp region Tm=60C, whole=76C) 5'- AATGATACGGCGACCACCGAGATCTACAC[i5]GGGAGATCGTCGGCAGCGTC -3' (i5XX_T7-1_long, comp region Tm=65C, whole=77C) 5β- AATGATACGGCGACCACCGAGATCTACAC[i5]TCGTCGGCAGCGTC -3' (i5XX_orig, comp region Tm=56C, whole=75C) XXXXXX DNA 5β- /5Phos/GGGAGATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG NNNNNNNNNNNN CTGTCTCTTATACACATCTCCGAGCCCACGAGAC -3' cDNA 3'- CCCTCTAGCAGCCGTCGCAGTCTACACATATTCTCTGTC NNNNNNNNNNNN GACAGAGAATATGTGTAGAGGCTCGGGTGCTCTG -5' (i7XX_orig, comp region Tm=57C, whole=73C) 3'- GGCTCGGGTGCTCTG[i7]TAGAGCATACGGCAGAAGACGAAC -5' (i7XX_long, comp region Tm=59C, whole=73C) 3'- GAGGCTCGGGTGCTCTG[i7]TAGAGCATACGGCAGAAGACGAAC -5' (i7_PCR_ILAdptr, Tm=58C) 3'- AGCATACGGCAGAAGACGAAC -5' '''PCR barcode addition primers:''' '''Using i5_shrt or i5_long and i7_orig or i7_long, test to validate''' '''Final sequecing ready fragment''' nXTv2_i5_index_read -----> nXTv2_read1 -----> nXTv2_i7_index_read -----> 5'- AATGATACGGCGACCACCGAGATCTACAC -3' 5'- TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG -3' 5'- CTGTCTCTTATACACATCTCCGAGCCCACGAGAC -3' 5'- AATGATACGGCGACCACCGAGATCTACAC[i5]GGGAGATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG NNNNNNNNNNNN CTGTCTCTTATACACATCTCCGAGCCCACGAGAC[i7]ATCTCGTATGCCGTCTTCTGCTTG -3' 3'- TTACTATGCCGCTGGTGGCTCTAGATGTG[i5]CCCTCTAGCAGCCGTCGCAGTCTACACATATTCTCTGTC NNNNNNNNNNNN GACAGAGAATATGTGTAGAGGCTCGGGTGCTCTG[i7]TAGAGCATACGGCAGAAGACGAAC -5' 3'- GACAGAGAATATGTGTAGAGGCTCGGGTGCTCTG -5' <----- nXTv2_read2 sequences to order: T7-nXTv2_i5-1-top 5'- CATGGAATTAATACGACTCACTATAGGGAGATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG -3β T7-nXTv2_i5-2-top 5'- CATGGAATTAATACGACTCACTATAGGGAGACTCCTTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG -3β P-T7tspn-bot 5'- /5Phos/CTGTCTCTTATACACATCT -3' P-sss_nXTv2_prmr 5β- /5Phos/GGGAGATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG -3' nXTv2_i7_top 5β- GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG -3' P-nXTv2_i7_bot 5'- /5Phos/CTGTCTCTTATACACATCT -3' i5_PCR_ILAdptr 5'- AATGATACGGCGACCACCGA -3' i7_PCR_ILAdptr 5'- CAAGCAGAAGACGGCATACGA -3' i502_T7-1_shrt 5'- AATGATACGGCGACCACCGAGATCTACACCTCTCTATGGGAGATCGTCGGCAGC -3' i503_T7-1_shrt 5'- AATGATACGGCGACCACCGAGATCTACACTATCCTCTGGGAGATCGTCGGCAGC -3' i505_T7-1_shrt 5'- AATGATACGGCGACCACCGAGATCTACACGTAAGGAGGGGAGATCGTCGGCAGC -3' i506_T7-1_shrt 5'- AATGATACGGCGACCACCGAGATCTACACACTGCATAGGGAGATCGTCGGCAGC -3' i502_T7-1_long 5'- AATGATACGGCGACCACCGAGATCTACACCTCTCTATGGGAGATCGTCGGCAGCGTC -3' i503_T7-1_long 5'- AATGATACGGCGACCACCGAGATCTACACTATCCTCTGGGAGATCGTCGGCAGCGTC -3' i505_T7-1_long 5'- AATGATACGGCGACCACCGAGATCTACACGTAAGGAGGGGAGATCGTCGGCAGCGTC -3' i506_T7-1_long 5'- AATGATACGGCGACCACCGAGATCTACACACTGCATAGGGAGATCGTCGGCAGCGTC -3' i701_long 5'- CAAGCAGAAGACGGCATACGAGATTCGCCTTAGTCTCGTGGGCTCGGAG -3' i702_long 5'- CAAGCAGAAGACGGCATACGAGATCTAGTACGGTCTCGTGGGCTCGGAG -3' i703_long 5'- CAAGCAGAAGACGGCATACGAGATTTCTGCCTGTCTCGTGGGCTCGGAG -3' i704_long 5'- CAAGCAGAAGACGGCATACGAGATGCTCAGGAGTCTCGTGGGCTCGGAG -3' sequencing primers used: nXTv2_i5_index 5'- AATGATACGGCGACCACCGAGATCTACAC -3' nXTv2_i7_index 5'- CTGTCTCTTATACACATCTCCGAGCCCACGAGAC -3' nXTv2_read1 5'- TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG -3' nXTv2_read2 5'- GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG -3' '''Modified Nextera XT V2 i5 barcode sequences and adaptors''' num. i5_barcode i5_name i5_smplsheet i5_full_sequence_(5'_->_3') 1 CTCTCTAT S502_T7-1_shrt CTCTCTAT AATGATACGGCGACCACCGAGATCTACACCTCTCTATGGGAGATCGTCGGCAGC 2 TATCCTCT S503_T7-1_shrt TATCCTCT AATGATACGGCGACCACCGAGATCTACACTATCCTCTGGGAGATCGTCGGCAGC 3 GTAAGGAG S505_T7-1_shrt GTAAGGAG AATGATACGGCGACCACCGAGATCTACACGTAAGGAGGGGAGATCGTCGGCAGC 4 ACTGCATA S506_T7-1_shrt ACTGCATA AATGATACGGCGACCACCGAGATCTACACACTGCATAGGGAGATCGTCGGCAGC 5 AAGGAGTA S507_T7-1_shrt AAGGAGTA AATGATACGGCGACCACCGAGATCTACACAAGGAGTAGGGAGATCGTCGGCAGC 6 CTAAGCCT S508_T7-1_shrt CTAAGCCT AATGATACGGCGACCACCGAGATCTACACCTAAGCCTGGGAGATCGTCGGCAGC 7 CGTCTAAT S510_T7-1_shrt CGTCTAAT AATGATACGGCGACCACCGAGATCTACACCGTCTAATGGGAGATCGTCGGCAGC 8 TCTCTCCG S511_T7-1_shrt TCTCTCCG AATGATACGGCGACCACCGAGATCTACACTCTCTCCGGGGAGATCGTCGGCAGC 9 TCGACTAG S513_T7-1_shrt TCGACTAG AATGATACGGCGACCACCGAGATCTACACTCGACTAGGGGAGATCGTCGGCAGC 10 TTCTAGCT S515_T7-1_shrt TTCTAGCT AATGATACGGCGACCACCGAGATCTACACTTCTAGCTGGGAGATCGTCGGCAGC 11 CCTAGAGT S516_T7-1_shrt CCTAGAGT AATGATACGGCGACCACCGAGATCTACACCCTAGAGTGGGAGATCGTCGGCAGC 12 GCGTAAGA S517_T7-1_shrt GCGTAAGA AATGATACGGCGACCACCGAGATCTACACGCGTAAGAGGGAGATCGTCGGCAGC 13 CTATTAAG S518_T7-1_shrt CTATTAAG AATGATACGGCGACCACCGAGATCTACACCTATTAAGGGGAGATCGTCGGCAGC 14 AAGGCTAT S520_T7-1_shrt AAGGCTAT AATGATACGGCGACCACCGAGATCTACACAAGGCTATGGGAGATCGTCGGCAGC 15 GAGCCTTA S521_T7-1_shrt GAGCCTTA AATGATACGGCGACCACCGAGATCTACACGAGCCTTAGGGAGATCGTCGGCAGC 16 TTATGCGA S522_T7-1_shrt TTATGCGA AATGATACGGCGACCACCGAGATCTACACTTATGCGAGGGAGATCGTCGGCAGC '''original Nextera XT V2 i5/i7 barcode sequencers and primers''' num. i5_barcode i5_name i5_smplsheet i5_full_sequence 1 CTCTCTAT S502 CTCTCTAT AATGATACGGCGACCACCGAGATCTACACCTCTCTATTCGTCGGCAGCGTC 2 TATCCTCT S503 TATCCTCT AATGATACGGCGACCACCGAGATCTACACTATCCTCTTCGTCGGCAGCGTC 3 GTAAGGAG S505 GTAAGGAG AATGATACGGCGACCACCGAGATCTACACGTAAGGAGTCGTCGGCAGCGTC 4 ACTGCATA S506 ACTGCATA AATGATACGGCGACCACCGAGATCTACACACTGCATATCGTCGGCAGCGTC 5 AAGGAGTA S507 AAGGAGTA AATGATACGGCGACCACCGAGATCTACACAAGGAGTATCGTCGGCAGCGTC 6 CTAAGCCT S508 CTAAGCCT AATGATACGGCGACCACCGAGATCTACACCTAAGCCTTCGTCGGCAGCGTC 7 CGTCTAAT S510 CGTCTAAT AATGATACGGCGACCACCGAGATCTACACCGTCTAATTCGTCGGCAGCGTC 8 TCTCTCCG S511 TCTCTCCG AATGATACGGCGACCACCGAGATCTACACTCTCTCCGTCGTCGGCAGCGTC 9 TCGACTAG S513 TCGACTAG AATGATACGGCGACCACCGAGATCTACACTCGACTAGTCGTCGGCAGCGTC 10 TTCTAGCT S515 TTCTAGCT AATGATACGGCGACCACCGAGATCTACACTTCTAGCTTCGTCGGCAGCGTC 11 CCTAGAGT S516 CCTAGAGT AATGATACGGCGACCACCGAGATCTACACCCTAGAGTTCGTCGGCAGCGTC 12 GCGTAAGA S517 GCGTAAGA AATGATACGGCGACCACCGAGATCTACACGCGTAAGATCGTCGGCAGCGTC 13 CTATTAAG S518 CTATTAAG AATGATACGGCGACCACCGAGATCTACACCTATTAAGTCGTCGGCAGCGTC 14 AAGGCTAT S520 AAGGCTAT AATGATACGGCGACCACCGAGATCTACACAAGGCTATTCGTCGGCAGCGTC 15 GAGCCTTA S521 GAGCCTTA AATGATACGGCGACCACCGAGATCTACACGAGCCTTATCGTCGGCAGCGTC 16 TTATGCGA S522 TTATGCGA AATGATACGGCGACCACCGAGATCTACACTTATGCGATCGTCGGCAGCGTC num. i7_barcode i7_name i7_smplsheet i7_full_sequence 1 TCGCCTTA N701 TAAGGCGA CAAGCAGAAGACGGCATACGAGATTCGCCTTAGTCTCGTGGGCTCGG 2 CTAGTACG N702 CGTACTAG CAAGCAGAAGACGGCATACGAGATCTAGTACGGTCTCGTGGGCTCGG 3 TTCTGCCT N703 AGGCAGAA CAAGCAGAAGACGGCATACGAGATTTCTGCCTGTCTCGTGGGCTCGG 4 GCTCAGGA N704 TCCTGAGC CAAGCAGAAGACGGCATACGAGATGCTCAGGAGTCTCGTGGGCTCGG 5 AGGAGTCC N705 GGACTCCT CAAGCAGAAGACGGCATACGAGATAGGAGTCCGTCTCGTGGGCTCGG 6 CATGCCTA N706 TAGGCATG CAAGCAGAAGACGGCATACGAGATCATGCCTAGTCTCGTGGGCTCGG 7 GTAGAGAG N707 CTCTCTAC CAAGCAGAAGACGGCATACGAGATGTAGAGAGGTCTCGTGGGCTCGG 8 CAGCCTCG N710 CGAGGCTG CAAGCAGAAGACGGCATACGAGATCAGCCTCGGTCTCGTGGGCTCGG 9 TGCCTCTT N711 AAGAGGCA CAAGCAGAAGACGGCATACGAGATTGCCTCTTGTCTCGTGGGCTCGG 10 TCCTCTAC N712 GTAGAGGA CAAGCAGAAGACGGCATACGAGATTCCTCTACGTCTCGTGGGCTCGG 11 TCATGAGC N714 GCTCATGA CAAGCAGAAGACGGCATACGAGATTCATGAGCGTCTCGTGGGCTCGG 12 CCTGAGAT N715 ATCTCAGG CAAGCAGAAGACGGCATACGAGATCCTGAGATGTCTCGTGGGCTCGG 13 TAGCGAGT N716 ACTCGCTA CAAGCAGAAGACGGCATACGAGATTAGCGAGTGTCTCGTGGGCTCGG 14 GTAGCTCC N718 GGAGCTAC CAAGCAGAAGACGGCATACGAGATGTAGCTCCGTCTCGTGGGCTCGG 15 TACTACGC N719 GCGTAGTA CAAGCAGAAGACGGCATACGAGATTACTACGCGTCTCGTGGGCTCGG 16 AGGCTCCG N720 CGGAGCCT CAAGCAGAAGACGGCATACGAGATAGGCTCCGGTCTCGTGGGCTCGG 17 GCAGCGTA N721 TACGCTGC CAAGCAGAAGACGGCATACGAGATGCAGCGTAGTCTCGTGGGCTCGG 18 CTGCGCAT N722 ATGCGCAG CAAGCAGAAGACGGCATACGAGATCTGCGCATGTCTCGTGGGCTCGG 19 GAGCGCTA N723 TAGCGCTC CAAGCAGAAGACGGCATACGAGATGAGCGCTAGTCTCGTGGGCTCGG 20 CGCTCAGT N724 ACTGAGCG CAAGCAGAAGACGGCATACGAGATCGCTCAGTGTCTCGTGGGCTCGG 21 GTCTTAGG N726 CCTAAGAC CAAGCAGAAGACGGCATACGAGATGTCTTAGGGTCTCGTGGGCTCGG 22 ACTGATCG N727 CGATCAGT CAAGCAGAAGACGGCATACGAGATACTGATCGGTCTCGTGGGCTCGG 23 TAGCTGCA N728 TGCAGCTA CAAGCAGAAGACGGCATACGAGATTAGCTGCAGTCTCGTGGGCTCGG 24 GACGTCGA N729 TCGACGTC CAAGCAGAAGACGGCATACGAGATGACGTCGAGTCTCGTGGGCTCGG '''UPDATE 8/27/2015''' [http://genome-tech.ucsd.edu/LabNotes/index.php/Brandon:LabNotes/Project1/2015-8-27 8/27/2015 T7-nXTv2_i5-1 testing]
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