Brandon:LabNotes/Project1/2013-7-10
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Testing new method of RNA processing, No RNase III, No polyA polymerase (PAP)
- In performing the Tn5 accessibility method, the most troubling steps are when RNase III is used for fragmentation and polyA polymerase is used to add polyA tails to the 3' end, so the T20VN_PE_R primer can be used to add adaptors/barcodes etc. Thus this protocol avoids the usage of both enzymes. RNase III does not seem to have a good shelf life and does not function correctly if it is not taken care of, and stops working approximately 3 months after purchase. PAP is a extremely robust enzyme, and adds polyA to most segments. Most of the unmapped reads are polyA tailed fragments with the sequencing reads consisting of polyA sequences.
- Changes & testing:
- Another robust method of generating cDNA will be used, using random hexamers for priming and MMLV for RT
- RNA in the RNA/cDNA hybrid will be degraged with RNase H, and heated for denaturing. Next, extension with BST 2.0 or Klenow exo- will be done with a DNA primer to the 5' end.
- DNA will will need to be fragmented, but this time a custom transpsome will be created that only has the nextera B ends on it, the 3' end of fragments were barcodes are attached.
- BST, taq2x, klenow exo- and DNAPI will be tested for second strand synthesis, with and without Rnase H treatment.
- In the end Fragmentation is still needed and is being performed by Nextera transposition. Other ideas for DNA fragmentation could also be entertained.
Sequences information, Using T7tspn-top2
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, 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) RNA 5’- GGGAGATCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNNNNN... -3' After single strand cDNA synthesis to form cDNA/RNA hybrid RNA 5’- GGGAGATCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNNNNN... -3' cDNA 3'- CCCTCTAGGAGGGAGCGCGGTAGTCTCTACACATATTCTCTGTC NNNNNNNNNNNN... -5' After Rnase H nicking, denatuation of small RNAs? and addition of 5' end primer. Extenstion with BST or klenow exo- or DNAP1 or Taq2x DNA /5Phos/GGGAGATCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG -3'(Tm=79) (sss_NPA_prmr) (No Poly A) cDNA 3'- CCCTCTAGGAGGGAGCGCGGTAGTCTCTACACATATTCTCTGTC NNNNNNNNNNNN... -5' Now have double stranded DNA, which should be long. DNA /5Phos/GGGAGATCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNNNNN... -3' cDNA 3'- CCCTCTAGGAGGGAGCGCGGTAGTCTCTACACATATTCTCTGTC NNNNNNNNNNNN... -5'
normal Nextera 5' end: 5'-GCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG-3' ("A-METS" NXTA_ME_A) 3'- TCTACACATATTCTCTGTC/5Phos/ (NXTA_ME_BOT, "pMENTS") after fill in: 5'- GCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNNNN -3' 3'- CGGAGGGAGCGCGGTAGTCTCTACACATATTCTCTGTC NNNNNNNNNNN -5' PCR amplification: 5'-AATGATACGGCGACCACCGA-3' (NXTA_P1) 5'-AATGATACGGCGACCACCGAGATCTACACGCCTCCCTCGCGCCATCAG-3' (NXTA_adpt1) 5'- GCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNNNN -3' 3'- CGGAGGGAGCGCGGTAGTCTCTACACATATTCTCTGTC NNNNNNNNNNN -5'
Custom Nextera transposome 5'- /5Phos/CTGTCTCTTATACACATCT -3' (NXTA_ME_BOT, "pMENTS") 3'- GACAGAGAATATGTGTAGAGACTCGCCCGACCGTTCCG -5' (NXTA_ME_B, "B-METS") 3'- TCTACACATATTCTCTGTC/5Phos/ (NXTA_ME_BOT, "pMENTS") 5'- GCCTTGCCAGCCCGCTCAGAGATGTGTATAAGAGACAG -3' (NXTA_ME_B, "B-METS") After custom nextera tagmentation: DNA /5Phos/GGGAGATCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNNNNN CTGTCTCTTATACACATCT -3' cDNA 3'- CCCTCTAGGAGGGAGCGCGGTAGTCTCTACACATATTCTCTGTC NNNNNNNNNNNN GACAGAGAATATGTGTAGAGACTCGCCCGACCGTTCCG -5' After fill in: DNA /5Phos/GGGAGATCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNNNNN CTGTCTCTTATACACATCTCTGAGCGGGCTGGCAAGGC -3' cDNA 3'- CCCTCTAGGAGGGAGCGCGGTAGTCTCTACACATATTCTCTGTC NNNNNNNNNNNN GACAGAGAATATGTGTAGAGACTCGCCCGACCGTTCCG -5' Amplification: 5’- [AATGATACGGCGACCACCGA]GGG]AGATCCTCCCTCGCGCCATCAGAG T7-top2-PCR-iaf3 (comp part (Tm=77), all (Tm=85C) 5’- [AATGATACGGCGACCACCGA]AGATCCTCCCTCGCGCCATCAGAG T7-top2-PCR-iaf2 (comp part (Tm=73), all (Tm=83C)) X X 5’- [AATGATACGGCGACCACCGA]GATCTCTCCCTCGCGCCATCAGAGAT T7-top2-PCR-iaf (original) (comp region 70C) (whole Tm=83C) X X XXXXX DNA /5Phos/GGG]AGATCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNNNNN CTGTCTCTTATACACATCTCTGAGCGGGCTGGCAAGGC -3' cDNA 3'- CCC]TCTAGGAGGGAGCGCGGTAGTCTCTACACATATTCTCTGTC NNNNNNNNNNNN GACAGAGAATATGTGTAGAGACTCGCCCGACCGTTCCG -5' 3'- GACTCGCCCGACCGTTCCGTCTGGC[GACACA]TAGAGCATACGGCAGAAGACGAAC -5' (custom barcodes) (Tm=86C) 3'- GACTCGCCCGACCGTTCCGTCTGGC[------]TAGAGCATACGGCAGAAGACGAAC -5' (NXTA_adpt2)(comp region Tm=73C) (whole Tm=85C) 3'- AGCATACGGCAGAAGACGAAC -5' (NXTA_P2)
buffers compatibility
- Clontech MMLV
Invitrogen 5X First-Strand Buffer 250 mM Tris-HCl (pH 8.3 at room temperature 375 mM KCl 15 mM MgCl2 0.1 M DTT Storage buffer 20 mM Tris-HCl (pH 7.5) 100 mM NaCl 0.1 mM EDTA 1 mM DTT 0.01% (v/v) NP-40 50% (v/v) glycerol
- NEB BST 2.0 Polymerase
1X Isothermal Amplification Buffer Pack: 20 mM Tris-HCl 10 mM (NH4)2SO4 50 mM KCl 2 mM MgSO4 0.1% Tween® 20 pH 8.8 @ 25°C Storage Temperature -20°C Storage Conditions 10 mM Tris-HCl 50 mM KCl 1 mM DTT 0.1 mM EDTA 50% Glycerol 0.1% Triton® X-100 pH 7.5 @ 25°C Heat Inactivation 80°C for 20 min
- NEB taq2x
Storage Temperature -20°C Buffer Composition 10 mM Tris-HCl 50 mM KCl 1.5 mM MgCl2 0.2 mM dNTPs 5% Glycerol 0.08% IGEPAL® CA-630 0.05% Tween® 20 25 units/ml Taq DNA Polymerase pH 8.6@25°C
- Epicentre klenow exo-
Storage Buffer: 50% glycerol solution 50 mM Tris-HCl (pH 7.5) 100 mM NaCl 1.0 mM dithiothreitol (DTT) 0.1 mM EDTA 0.1% Triton® X-100 10X reaction buffer 0.2 M Tris-HCl (pH 7.5) 50 mM MgCl2 5.0 mM DTT.
- Invitrogen DNAPI
Unit Reaction Conditions: 50 mM potassium phosphate (pH 7.0) 6.7 mM MgCl2 1 mM 2-mercaptoethanol 80 µg/ml templateprimer 32 µM dTTP 69 nM [3H]dTTP and enzyme in 100 µl for 30 min. at 37°C. NEB DNAPI 1X NEBuffer 2: 50 mM NaCl 10 mM Tris-HCl 10 mM MgCl2 1 mM DTT pH 7.9 @ 25°C Heat Inactivation 75°C for 20 min
- RNase H
Supplied in 20 mM Tris-HCl 100 mM KCl 10 mM MgCl2 0.1 mM EDTA 0.1 mM DTT 50% glycerol pH 7.9 @ 25°C 10X RNAse H Buffer (B9220): 500 mM Tris-HCl 750 mM KCl 30 mM MgCl2 100 mM DTT pH 8.3 @ 25°C
- nextera
5x Tagmentation reaction buffer 50 mM Tris-OAc pH 8.0 25 mM Mg(OAc)2
Protocol
- check is have reagents etc
1. generating custom nextera transposome
2. polymerase conditions:
Taq2X Incubate at 72C for 8 minutes (Tm of DNA primer is 79C) (no reaction buffer) Klenow exo- 30 minutes at 37°C un (10X reaction buffer) BST polymerase: (10X reaction buffer) Reaction Conditions 1X Isothermal Amplification Buffer Incubate at 65°C (for 30 mintues) DNAP (no reaction buffer right now) in 30 min. at 37°C Rnase H (10X reaction buffer) 20 minutes at 37°C Heat Inactivation 65°C for 20 min