Brandon:LabNotes/Project1/2012-5-7: Difference between revisions
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*would result in 100% efficiency of capture of sequence upon insertion, including single insertions. | *would result in 100% efficiency of capture of sequence upon insertion, including single insertions. | ||
Summary: | ===Summary for using T7tspn-top:=== | ||
Sequences: | Sequences: | ||
5’- [A][TTAATACGACTCACTATAGGG][T][CCTCCCTCGCGCCATCA][G][AGATGTGTATAAGAGACAG] -3’ (T7tspn-top) | |||
spcr T7 forward spcr read sequence spcr ME | |||
5'- [phos]CTGTCTCTTATACACATCT -3' (T7tspn-bot) | 5'- [phos]CTGTCTCTTATACACATCT -3' (T7tspn-bot) | ||
T7 description: | |||
+1 | |||
CATGAGA[TTAATACGACTCACTATA|G|GGAGA] | |||
[end sequence for optimal binding] T7 --> [front sequence for optimal binding] | |||
Line 52: | Line 53: | ||
After IVT. Addition of PolyA with E. coli PolyA polymerase (Ambion kit). All RNA strands will have same 5' end. | 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 5’- GGGTCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNNNNN... -3' | |||
RNA 5’- GGGTCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNNNNN AAAAAAAAAAAAAAAAAAAAAAAA -3' | |||
Adaptors to add for first/second strand cDNA synthesis. | |||
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 | |||
5’- [AATGATACGGCGACCACCGA][GATCT][CTCCCTCGCGCCATCAGAGAT] -3’ (Tm=66.79 comp region only) | |||
XXXX | |||
RNA 5’- GGGTC][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][GAACAT][TAGAGCATACGGCAGAAGACGAAC] -5' | |||
adaptor2 Tm=64.41 spr bc ILA adaptor orange | |||
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 | |||
5’- [AATGATACGGCGACCACCGA][GATCT][CTCCCTCGCGCCATCAGAGAT] -3’(Tm=66.79 comp region only) | |||
XXXX | |||
RNA 5’- GGGTC][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 | |||
===Summary for using with T7tspn-top2: (USED)=== | |||
Sequences: | |||
5'- [CATGAGA][TTAATACGACTCACTATAG][GGAGA][TCCTCCCTCGCGCCATCA][G][AGATGTGTATAAGAGACAG] -3’ (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: | |||
5’- CATGAGATTAATACGACTCACTATAGGGAGATCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG -3’ (T7tspn-top2) | |||
3'- TCTACACATATTCTCTGTC [Phos]-5' (T7tspn-bot) | |||
5'- [phos] CTGTCTCTTATACACATCT -3' | |||
3’- GACAGAGAATATGTGTAGAGACTACCGCGCTCCCTCCTAGAGGGATATCACTCAGCATAATTAGAGTAC -5’ | |||
Fragments created after transposition: | |||
5’- CATGAGA[TTAATACGACTCACTATAGGGAGA]TCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNN------ CTGTCTCTTATACACATCT -3' | |||
3'- TCTACACATATTCTCTGTC ------NNNNNN GACAGAGAATATGTGTAGAGACTACCGCGCTCCCTCCT[AGAGGGATATCACTCAGCATAATT]AGAGTAC -5’ | |||
Upon single insertion, T7 still present in either direction of insertion, IVT off top strand, and bottom: | |||
NNNNN------ CTGTCTCTTATACACATCT -3' 5’- CATGAGA[TTAATACGACTCACTATAGGGAGA]TCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNNN | |||
NNNNNNNNNNN GACAGAGAATATGTGTAGAGACTACCGCGCTCCCTCCT[AGAGGGATATCACTCAGCATAATT]AGAGTAC -5’ 3'- TCTACACATATTCTCTGTC ------NNNN | |||
After fill in with taq polymerase (2X) (could use BST, T4 DNA poly also) | |||
5’- CATGAGA[TTAATACGACTCACTATAGGGAGA]TCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNNNNN | |||
3'- GTACTCT[AATTATGCTGAGTGATATCCCTCT]AGGAGGGAGCGCGGTAGTCTCTACACATATTCTCTGTC (NNNNNN)NNNN | |||
NNN(NNNNNN) CTGTCTCTTATACACATCTCTGATGGCGCGAGGGAGGA[TCTCCCTATAGTGAGTCGTATTAA]TCTCATG -3' | |||
NNNNNNNNNNN GACAGAGAATATGTGTAGAGACTACCGCGCTCCCTCCT[AGAGGGATATCACTCAGCATAATT]AGAGTAC -5’ | |||
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 5’- GGGAGATCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNNNNN... -3' | |||
RNA 5’- 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 | |||
5’- [AATGATACGGCGACCACCGA][GATCT][CTCCCTCGCGCCATCAGAGAT] -3’(Tm=66.79 comp region only) | |||
X X XXXXX | |||
RNA 5’- 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' | |||
5’- 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 | |||
5’- [AATGATACGGCGACCACCGA][GATCT][CTCCCTCGCGCCATCAGAGAT] -3’(Tm=66.79 comp region only) | |||
X X XXXXX | |||
RNA 5’- 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 | |||
5’- [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 | |||
===Summary for using with T7tspn-top3:=== | |||
Sequences: | |||
5'- [CATGAGA][TTAATACGACTCACTATAG][GGAGA][CATTA][AGATGTGTATAAGAGACAG] -3’ (T7tspn-top3) | |||
spcr T7 forward [req spcr] [rd spcr] ME | |||
5'- [phos]CTGTCTCTTATACACATCT -3' (T7tspn-bot) | |||
G][GGAGA][CATTA][AGATGTGTATAAGAGACAG (Tm 60.71) | |||
T7 description: | |||
+1 | |||
CATGAGA[TTAATACGACTCACTATA|G|GGAGA] | |||
[end sequence for optimal binding] T7 --> [front sequence for optimal binding] | |||
After Annealing: | |||
5'- CATGAGATTAATACGACTCACTATAGGGAGACATTAAGATGTGTATAAGAGACAG -3’ (T7tspn-top3) | |||
3'- TCTACACATATTCTCTGTC [Phos]-5' (T7tspn-bot) | |||
5'- [phos] CTGTCTCTTATACACATCT -3' | |||
3’- GACAGAGAATATGTGTAGAATTACAGAGGGATATCACTCAGCATAATTAGAGTAC -5’ | |||
Fragments created after transposition: | |||
5’- CATGAGA[TTAATACGACTCACTATAGGGAGA]CATTAAGATGTGTATAAGAGACAG NNNNNN------ CTGTCTCTTATACACATCT -3' | |||
3'- TCTACACATATTCTCTGTC ------NNNNNN GACAGAGAATATGTGTAGAATTAC[AGAGGGATATCACTCAGCATAATT]AGAGTAC -5’ | |||
Upon single insertion, T7 still present in either direction of insertion, IVT off top strand, and bottom: | |||
NNNNN------ CTGTCTCTTATACACATCT -3' 5’- CATGAGA[TTAATACGACTCACTATAGGGAGA]CATTAAGATGTGTATAAGAGACAG NNNNNNNNNN | |||
NNNNNNNNNNN GACAGAGAATATGTGTAGAATTAC[AGAGGGATATCACTCAGCATAATT]AGAGTAC -5’ 3'- TCTACACATATTCTCTGTC ------NNNN | |||
After fill in with taq polymerase (2X) (could use BST, T4 DNA poly also) | |||
5’- CATGAGA[TTAATACGACTCACTATAGGGAGA]CATTAAGATGTGTATAAGAGACAG NNNNNNNNNN | |||
3'- GTACTCT[AATTATGCTGAGTGATATCCCTCT]GTAATTCTACACATATTCTCTGTC (NNNNNN)NNNN | |||
NNN(NNNNNN) CTGTCTCTTATACACATCTTAATG[TCTCCCTATAGTGAGTCGTATTAA]TCTCATG -3' | |||
NNNNNNNNNNN GACAGAGAATATGTGTAGAATTAC[AGAGGGATATCACTCAGCATAATT]AGAGTAC -5’ | |||
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 5’- GGGAGACATTAAGATGTGTATAAGAGACAG NNNNNNNNNN -3' | |||
5’- | RNA 5’- GGGAGACATTAAGATGTGTATAAGAGACAG NNNNNNNNNN AAAAAAAAAAAAAAAAAAAAAAAA -3' | ||
Adaptors for first/second strand cDNA synthesis, and addition of lawn adaptors | |||
POLY-A METHOD | |||
Adaptors for first/second strand cDNA synthesis, and addition of lawn adaptors | |||
(second strand synthesis) | |||
(Tm=60.71 comp region only) | |||
ILA adaptor blue (T7-top3-PCR-iaf) Tm=81.40 | |||
5'- [AATGATACGGCGACCACCGA][GATCT][GGGAGACATTAAGATGTGTATAAGAGACAG] -3' | |||
RNA 5’- GGGAGACATTAAGATGTGTATAAGAGACAG NNNNNNNNNN 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][GAACAT][TAGAGCATACGGCAGAAGACGAAC] -5' | |||
adaptor2 Tm=64.41 spr bc ILA adaptor orange | |||
RANDOM NONAMER METHOD | |||
Adaptors for first/second strand cDNA synthesis, and addition of lawn adaptors | |||
(second strand synthesis) | |||
(Tm=60.71 comp region only) | |||
ILA adaptor blue (T7-top3-PCR-iaf) Tm=81.40 | |||
5'- [AATGATACGGCGACCACCGA][GATCT][GGGAGACATTAAGATGTGTATAAGAGACAG] -3' | |||
RNA 5’- GGGAGACATTAAGATGTGTATAAGAGACAG NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN -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 | |||
Line 144: | Line 369: | ||
9. addition of | 9. addition of adaptors to 3' ends and PCR amplification | ||
10. size selection. | 10. size selection. | ||
===ordered=== | ===ordered/to order=== | ||
(T7tspn-top) 5’- ATTAATACGACTCACTATAGGGTCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG -3’ | (T7tspn-top) 5’- ATTAATACGACTCACTATAGGGTCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG -3’ | ||
(T7tspn-bot) 5'- /5phos/CTGTCTCTTATACACATCT -3' | (T7tspn-bot) 5'- /5phos/CTGTCTCTTATACACATCT -3' | ||
For using with (T7tspn-top) | |||
(T7tspn-top2) 5'- | |||
(T7tspn-read1) 5'- TCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG -3' | |||
(T7-sss-1) 5'- [AATGATACGGCGACCACCGAGATCTACACGCCT]GGGTCCTCCCTCGCGCCATCAGAGA -3' | |||
For using with(T7-top2) | |||
Need 5+ NT upstream of T7 for max efficiency | |||
(T7-top2) 5’- CATGAGATTAATACGACTCACTATAGGGAGATCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG -3’ | |||
(T7-fss) 3'- V TTTTTTTTTTTTTTTTTTTTTTTTCTGGC[bc][TAGAGCATACGGCAGAAGACGAAC] -5' | |||
(T7-fss-shrt) 3'- V TTTTTTTTTTTTTTTTTTTTTTTT -5' | |||
(T7-sss-2) 5'-[AATGATACGGCGACCACCGAGATCTACACGCCT]GGGAGATCCTCCCTCGCGCCATCAGAGA -3' | |||
(T7-Nprimer1) 5'- AATGATACGGCGACCACCGA -3' | |||
(T7-Nprimer2) 3'- AGCATACGGCAGAAGACGAAC -5' | |||
(T7tspn-Read1-2) 5'- AGATCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG -3' | |||
(T7tspn-Read2) 3'-V TTTTTTTTTTTTTTTTTTTTTTTTCTGGC -5' | |||
(T7tspn-indx) 5'- TTTTTTTTTTTTTTTTTCTGGC -3' | |||
quick and dirty validation of IVT RNA fragments with RT-PCR, For using with(T7-top2) and (T7tspn-top). | |||
(tspn-vldte-RT) (add on unique sequence to end as primer for PCR, since both ends are the same) | |||
5'- [CAAGCAGAAGACGGCATACGAGAT]GATGTGTATAAGAGACAG -3' | |||
(illuminia bPCR adaptor) ME | |||
(tspn-vldate-R) | |||
5'- CAAGCAGAAGACGGCATACGA -3' (primer 2, from nextera) | |||
(tspn-vldte-F) | |||
5'- TCCTCCCTCGCGCCATCAGAGAT -3' | |||
===left over notes=== | ===left over notes=== | ||
Second | Second adaptor to anneal: | ||
[phos] -5' CTGTCTCTTATACACATCTCTGATGGAGCGGGCTGGCAAGGCAGACCGATC -3’ | [phos] -5' CTGTCTCTTATACACATCTCTGATGGAGCGGGCTGGCAAGGCAGACCGATC -3’ |
Latest revision as of 23:49, 11 February 2014
Adaptation of transposition method for inserting T7[edit]
- in reference to shendure paper transposition
- also in reference to Athurva's summary
- new technique of only annealing sequences for form full mosaic ends and adding adaptors.
- would result in 100% efficiency of capture of sequence upon insertion, including single insertions.
Summary for using T7tspn-top:[edit]
Sequences: 5’- [A][TTAATACGACTCACTATAGGG][T][CCTCCCTCGCGCCATCA][G][AGATGTGTATAAGAGACAG] -3’ (T7tspn-top) spcr T7 forward 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: 5’- ATTAATACGACTCACTATAGGGTCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG -3’ 3'- TCTACACATATTCTCTGTC [Phos]-5' (T7tspn-bot) 5'- [phos] CTGTCTCTTATACACATCT -3' 3’- GACAGAGAATATGTGTAGAGACTACCGCGCTCCCTCCTGGGATATCACTCAGCATAATTA -5’ Fragments created after transposition: 5’- A[TTAATACGACTCACTATAGGG]TCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNN------ CTGTCTCTTATACACATC -3' 3'- CTACACATATTCTCTGTC ------NNNNNN GACAGAGAATATGTGTAGAGACTACCGCGCTCCCTCCT[GGGATATCACTCAGCATAATT]A -5’ Upon single insertion, T7 still present in either direction of insertion, IVT off top strand, and bottom: NNNNN------ CTGTCTCTTATACACATC -3' 5’- A[TTAATACGACTCACTATAGGG]TCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNNN NNNNNNNNNNN GACAGAGAATATGTGTAGAGACTACCGCGCTCCCTCCT[GGGATATCACTCAGCATAATT]A -5’ 3'- CTACACATATTCTCTGTC ------NNNN After fill in with taq polymerase (2X) (could use BST, T4 DNA poly also) 5’- A[TTAATACGACTCACTATAGGG]TCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNNNNN 3'- T[AATTATGCTGAGTGATATCCC]AGGAGGGAGCGCGGTAGTCTCTACACATATTCTCTGTC (NNNNNN)NNNN NNN(NNNNNN) CTGTCTCTTATACACATCTCTGATGGCGCGAGGGAGGA[CCCTATAGTGAGTCGTATTAA]T -3' NNNNNNNNNNN GACAGAGAATATGTGTAGAGACTACCGCGCTCCCTCCT[GGGATATCACTCAGCATAATT]A -5’ 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 5’- GGGTCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNNNNN... -3' RNA 5’- GGGTCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNNNNN AAAAAAAAAAAAAAAAAAAAAAAA -3' Adaptors to add for first/second strand cDNA synthesis. 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 5’- [AATGATACGGCGACCACCGA][GATCT][CTCCCTCGCGCCATCAGAGAT] -3’ (Tm=66.79 comp region only) XXXX RNA 5’- GGGTC][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][GAACAT][TAGAGCATACGGCAGAAGACGAAC] -5' adaptor2 Tm=64.41 spr bc ILA adaptor orange 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 5’- [AATGATACGGCGACCACCGA][GATCT][CTCCCTCGCGCCATCAGAGAT] -3’(Tm=66.79 comp region only) XXXX RNA 5’- GGGTC][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
Summary for using with T7tspn-top2: (USED)[edit]
Sequences: 5'- [CATGAGA][TTAATACGACTCACTATAG][GGAGA][TCCTCCCTCGCGCCATCA][G][AGATGTGTATAAGAGACAG] -3’ (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: 5’- CATGAGATTAATACGACTCACTATAGGGAGATCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG -3’ (T7tspn-top2) 3'- TCTACACATATTCTCTGTC [Phos]-5' (T7tspn-bot) 5'- [phos] CTGTCTCTTATACACATCT -3' 3’- GACAGAGAATATGTGTAGAGACTACCGCGCTCCCTCCTAGAGGGATATCACTCAGCATAATTAGAGTAC -5’ Fragments created after transposition: 5’- CATGAGA[TTAATACGACTCACTATAGGGAGA]TCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNN------ CTGTCTCTTATACACATCT -3' 3'- TCTACACATATTCTCTGTC ------NNNNNN GACAGAGAATATGTGTAGAGACTACCGCGCTCCCTCCT[AGAGGGATATCACTCAGCATAATT]AGAGTAC -5’ Upon single insertion, T7 still present in either direction of insertion, IVT off top strand, and bottom: NNNNN------ CTGTCTCTTATACACATCT -3' 5’- CATGAGA[TTAATACGACTCACTATAGGGAGA]TCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNNN NNNNNNNNNNN GACAGAGAATATGTGTAGAGACTACCGCGCTCCCTCCT[AGAGGGATATCACTCAGCATAATT]AGAGTAC -5’ 3'- TCTACACATATTCTCTGTC ------NNNN After fill in with taq polymerase (2X) (could use BST, T4 DNA poly also) 5’- CATGAGA[TTAATACGACTCACTATAGGGAGA]TCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNNNNN 3'- GTACTCT[AATTATGCTGAGTGATATCCCTCT]AGGAGGGAGCGCGGTAGTCTCTACACATATTCTCTGTC (NNNNNN)NNNN NNN(NNNNNN) CTGTCTCTTATACACATCTCTGATGGCGCGAGGGAGGA[TCTCCCTATAGTGAGTCGTATTAA]TCTCATG -3' NNNNNNNNNNN GACAGAGAATATGTGTAGAGACTACCGCGCTCCCTCCT[AGAGGGATATCACTCAGCATAATT]AGAGTAC -5’ 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 5’- GGGAGATCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNNNNN... -3' RNA 5’- 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 5’- [AATGATACGGCGACCACCGA][GATCT][CTCCCTCGCGCCATCAGAGAT] -3’(Tm=66.79 comp region only) X X XXXXX RNA 5’- 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' 5’- 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 5’- [AATGATACGGCGACCACCGA][GATCT][CTCCCTCGCGCCATCAGAGAT] -3’(Tm=66.79 comp region only) X X XXXXX RNA 5’- 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 5’- [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
Summary for using with T7tspn-top3:[edit]
Sequences: 5'- [CATGAGA][TTAATACGACTCACTATAG][GGAGA][CATTA][AGATGTGTATAAGAGACAG] -3’ (T7tspn-top3) spcr T7 forward [req spcr] [rd spcr] ME 5'- [phos]CTGTCTCTTATACACATCT -3' (T7tspn-bot) G][GGAGA][CATTA][AGATGTGTATAAGAGACAG (Tm 60.71) T7 description: +1 CATGAGA[TTAATACGACTCACTATA|G|GGAGA] [end sequence for optimal binding] T7 --> [front sequence for optimal binding] After Annealing: 5'- CATGAGATTAATACGACTCACTATAGGGAGACATTAAGATGTGTATAAGAGACAG -3’ (T7tspn-top3) 3'- TCTACACATATTCTCTGTC [Phos]-5' (T7tspn-bot) 5'- [phos] CTGTCTCTTATACACATCT -3' 3’- GACAGAGAATATGTGTAGAATTACAGAGGGATATCACTCAGCATAATTAGAGTAC -5’ Fragments created after transposition: 5’- CATGAGA[TTAATACGACTCACTATAGGGAGA]CATTAAGATGTGTATAAGAGACAG NNNNNN------ CTGTCTCTTATACACATCT -3' 3'- TCTACACATATTCTCTGTC ------NNNNNN GACAGAGAATATGTGTAGAATTAC[AGAGGGATATCACTCAGCATAATT]AGAGTAC -5’ Upon single insertion, T7 still present in either direction of insertion, IVT off top strand, and bottom: NNNNN------ CTGTCTCTTATACACATCT -3' 5’- CATGAGA[TTAATACGACTCACTATAGGGAGA]CATTAAGATGTGTATAAGAGACAG NNNNNNNNNN NNNNNNNNNNN GACAGAGAATATGTGTAGAATTAC[AGAGGGATATCACTCAGCATAATT]AGAGTAC -5’ 3'- TCTACACATATTCTCTGTC ------NNNN After fill in with taq polymerase (2X) (could use BST, T4 DNA poly also) 5’- CATGAGA[TTAATACGACTCACTATAGGGAGA]CATTAAGATGTGTATAAGAGACAG NNNNNNNNNN 3'- GTACTCT[AATTATGCTGAGTGATATCCCTCT]GTAATTCTACACATATTCTCTGTC (NNNNNN)NNNN NNN(NNNNNN) CTGTCTCTTATACACATCTTAATG[TCTCCCTATAGTGAGTCGTATTAA]TCTCATG -3' NNNNNNNNNNN GACAGAGAATATGTGTAGAATTAC[AGAGGGATATCACTCAGCATAATT]AGAGTAC -5’ 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 5’- GGGAGACATTAAGATGTGTATAAGAGACAG NNNNNNNNNN -3' RNA 5’- GGGAGACATTAAGATGTGTATAAGAGACAG NNNNNNNNNN AAAAAAAAAAAAAAAAAAAAAAAA -3' Adaptors for first/second strand cDNA synthesis, and addition of lawn adaptors POLY-A METHOD Adaptors for first/second strand cDNA synthesis, and addition of lawn adaptors (second strand synthesis) (Tm=60.71 comp region only) ILA adaptor blue (T7-top3-PCR-iaf) Tm=81.40 5'- [AATGATACGGCGACCACCGA][GATCT][GGGAGACATTAAGATGTGTATAAGAGACAG] -3' RNA 5’- GGGAGACATTAAGATGTGTATAAGAGACAG NNNNNNNNNN 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][GAACAT][TAGAGCATACGGCAGAAGACGAAC] -5' adaptor2 Tm=64.41 spr bc ILA adaptor orange RANDOM NONAMER METHOD Adaptors for first/second strand cDNA synthesis, and addition of lawn adaptors (second strand synthesis) (Tm=60.71 comp region only) ILA adaptor blue (T7-top3-PCR-iaf) Tm=81.40 5'- [AATGATACGGCGACCACCGA][GATCT][GGGAGACATTAAGATGTGTATAAGAGACAG] -3' RNA 5’- GGGAGACATTAAGATGTGTATAAGAGACAG NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN -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
Experiments needed to be done for proof of concept/procedures[edit]
- procotols from shendure paper, shendure paper transposition
1. annealing of ME sequence to T7 transposon sequence
- a. Make 100 uM stock solution of T7tspn-top and T7tspn-bot.
- b. Incubate 10 uL of each oligo (100uM) with 80 uL EB buffer at 95C for 2 minutes. Oligo's now at 10 uM in 100 uL.
- c. cool to RT at 0.1 C/s
2. transposome complex generation, run controls!!! (nextera normal, without transposon, without transposase, Jurkat DNA only, transposon only)
- add the below components into one tube and incubate for 20 minutes at RT
2.5 uL of annealed transposon 2.5 uL of 100% sterile glycerol 5.0 uL of Ez-TN5 transposase
- store at -20, is good for a year
3. transposition reaction
- add the below into one tube and incubate for 8 minutes at 55C.
1 uL nextera HMW buffer 1 uL genomic DNA at prepared quantities (1 ng) 2 uL Nuclease free water 1 uL prepared T7 transposomes (MAKE SURE TO ADD LAST) ___________ 5 uL total solution
method used in shendure paper: 4 uL nextera HMW buffer X uL genomic DNA at prepared quantities X uL Nuclease free water ______ 17.5 uL total solution add 2.5 uL of prepared transposomes
4. Protease digestion of transposase, protease inactivation
To each tube, add: 1 uL Qiagen Protease, for 5 uL reaction 1 uL of .5 for .1 AU final. (stock is 5 AU and diluted 10X. want .5 AU/uL final []) Incubate: 50 C 10 minutes, 70 C 20 minutes
5. Fill in reaction
- Add 6 uL 2X taq polymerase, run at 72C for 3 minutes. (same as nextera)
- IVT on filled in reaction and not filled in reaction in to show fill-in is working.
6. should now be ready for IVT.
- Look at Ambion's, life tech's and NEB's T7s. for which one to buy.
7. RNA fragmentation
- Magnesium fragmentation or Shearing, look at what is done in RNA fragmentation kits
8. PolyA polymerase and ATP to A-tail 3'-ends
9. addition of adaptors to 3' ends and PCR amplification
10. size selection.
ordered/to order[edit]
(T7tspn-top) 5’- ATTAATACGACTCACTATAGGGTCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG -3’ (T7tspn-bot) 5'- /5phos/CTGTCTCTTATACACATCT -3' For using with (T7tspn-top) (T7tspn-read1) 5'- TCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG -3' (T7-sss-1) 5'- [AATGATACGGCGACCACCGAGATCTACACGCCT]GGGTCCTCCCTCGCGCCATCAGAGA -3' For using with(T7-top2) Need 5+ NT upstream of T7 for max efficiency (T7-top2) 5’- CATGAGATTAATACGACTCACTATAGGGAGATCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG -3’ (T7-fss) 3'- V TTTTTTTTTTTTTTTTTTTTTTTTCTGGC[bc][TAGAGCATACGGCAGAAGACGAAC] -5' (T7-fss-shrt) 3'- V TTTTTTTTTTTTTTTTTTTTTTTT -5' (T7-sss-2) 5'-[AATGATACGGCGACCACCGAGATCTACACGCCT]GGGAGATCCTCCCTCGCGCCATCAGAGA -3' (T7-Nprimer1) 5'- AATGATACGGCGACCACCGA -3' (T7-Nprimer2) 3'- AGCATACGGCAGAAGACGAAC -5' (T7tspn-Read1-2) 5'- AGATCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG -3' (T7tspn-Read2) 3'-V TTTTTTTTTTTTTTTTTTTTTTTTCTGGC -5' (T7tspn-indx) 5'- TTTTTTTTTTTTTTTTTCTGGC -3' quick and dirty validation of IVT RNA fragments with RT-PCR, For using with(T7-top2) and (T7tspn-top). (tspn-vldte-RT) (add on unique sequence to end as primer for PCR, since both ends are the same) 5'- [CAAGCAGAAGACGGCATACGAGAT]GATGTGTATAAGAGACAG -3' (illuminia bPCR adaptor) ME (tspn-vldate-R) 5'- CAAGCAGAAGACGGCATACGA -3' (primer 2, from nextera) (tspn-vldte-F) 5'- TCCTCCCTCGCGCCATCAGAGAT -3'
left over notes[edit]
Second adaptor to anneal: [phos] -5' CTGTCTCTTATACACATCTCTGATGGAGCGGGCTGGCAAGGCAGACCGATC -3’ 5’- ATTAATACGACTCACTATAGGGTCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNN------ CTGTCTCTTATACACATC -3' X X XXX X XXXX X XX XX XX XX X XXXX X XXX X X 3'- CTACACATATTCTCTGTC ------NNNNNNN GACAGAGAATATGTGTAGAGACTACCGCGCTCCCTCCTGGGATATCACTCAGCATAATTA -5’ 3'- CTAGCCAGACGGAACGGTCGGGCGAGGTAGTCTCTACACATATTCTCTGTC [phos] -5' Strands after fill-in, ligation, separation top strand 5’- ATTAATACGACTCACTATAGGGTCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNNNNNN CTGTCTCTTATACACATCTCTGATGGAGCGGGCTGGCAAGGCAGACCGATC -3’ bottom strand 3'- CTAGCCAGACGGAACGGTCGGGCGAGGTAGTCTCTACACATATTCTCTGTC NNNNNNNNNNNNN GACAGAGAATATGTGTAGAGACTACCGCGCTCCCTCCTGGGATATCACTCAGCATAATTA -5’ to stop nonspecific amplification in shendure protocol 5’-[Phos]- CTGTCTCTTATACACATC -3’ [ddC - 3' to block ends]