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Assays of tagmentation activity of Tn5 on RNA/DNA hybrids
Last updated date: Jan 15, 2021 Views: 1102 Forks: 0
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1. Pre-preparation
1) 4x Tagment buffer
Final | ||
| 1M Tris-HCl(pH=7.5) | 40ul | 40mM |
| 1M MgCl2 | 20ul | 20mM |
| 50% PEG8000 | 640ul | 32% |
| 100% PEG200 | 200ul | 20% |
| H2O | 100ul | |
| Total | 1000ul |
2) Adapter preparation
| Tn5ME-A(10uM) | 10ul |
| Tn5MErev(10uM) | 10ul |
| Total | 20ul |
| Tn5ME-B(10uM) | 10ul |
| Tn5MErev(10uM) | 10ul |
| Total | 20ul |
94℃, 5min; -1℃/min; to 10℃.
Mix annealed adaptor A and B with a 1:1 ratio.
3) Tn5 transposome assembly
| Reagents | Volume |
| Tn5 (Vazyme, 500ng/ul) | 4ul |
| Annealed Adapter A/B | 7ul |
| Coupling Buffer | 39ul |
| Total | 50ul |
Mix well by gently pipetting up and down. Incubate for 1 hour at 30℃. Store at -20℃.
2. Reverse transcription
(1)
| Reagents | Volume |
| Purified total RNA (2-200ng) | 1.75ul |
| 50uM Oligo dT(23)VN | 0.25ul |
| 10mM dNTP mix | 0.25ul |
| Total | 2.25ul |
65℃，5min; chill on ice
(2)
| Reagents | Volume |
| 50% PEG8000 | 0.75ul |
| 5x First Strand buffer (Thermo) | 1ul |
| 0.1M DTT | 0.25ul |
| Superase-In(20U/ul) | 0.5ul |
| SS IV (Thermo) | 0.25ul |
| Total | 2.75ul |
55℃, 2h; 80℃, 10min; hold at 4℃
3. Tagmentation
(1)
| Reagents | Volume |
| RT product | 5ul |
| 4x Tagment Buffer | 5ul |
| Superase-In | 1ul |
| Assembled Tn5 transposome | 0.5-2ul |
| H2O | 7-8.5ul |
| Total | 20ul |
55℃, 30min; hold at 10℃;
(2) Add 5ul 0.2% SDS(final 0.04%), incubate for 5min at room temperature.
4. Gap filling & PCR
(1)
| Reagents | Volume |
| Tagmentation product | 25ul |
| Bst 3.0 DNA polymerase(8U/ul) | 2.4ul |
| NEBNext Q5 hotstart HiFi PCR mix | 30ul |
| Total | 57.4ul |
72℃, 15min;
95℃, 5min;
4℃, forever;
(2)
| N5* primer(10uM) | 1.3ul |
| N7* primer(10uM) | 1.3ul |
| Total | 2.6ul |
98℃, 30s;
65℃, 10min 4℃, forever
(3) Purified by 1X XP beads (60ul), eluted by 32ul H2O.
(4) Transfer 30ul to a new tube,purified by 1X XP beads(30ul) again, eluted by 10ul H2O.
(5) Transfer 8ul to a new tube, determine the concentration by the Qubit and assess the size distribution by the fragment analyzer.
200ng total RNA as input
20ng total RNA as input
2ng total RNA as input
- Lu, B, Dong, L, Yi, D, Zhang, M, Zhu, C, Li, X and Yi, C(2021). Assays of tagmentation activity of Tn5 on RNA/DNA hybrids. Bio-protocol Preprint. bio-protocol.org/prep753.
- Lu, B., Dong, L., Yi, D., Zhang, M., Zhu, C., Li, X. and Yi, C.(2020). Transposase-assisted tagmentation of RNA/DNA hybrid duplexes. eLife. DOI: 10.7554/eLife.54919
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