Method Article
Nascent RNA 4sU labelling and enrichment
https://doi.org/10.21203/rs.3.pex-1245/v1
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4sU-seq allows for measuring or nascent transcription which can reflect dynamic changes more accurately than traditional RNA-seq. Incorporation of the nucleoside analog 4-thiouridine into RNA allows the separation of nascent transcripts though reversible coupling to biotin and streptavdin bead pull-down. The protocol takes roughly two days to generate a sequencing ready library.
4sU-seq allows for measuring or nascent transcription which can reflect dynamic expression changes more accurately than traditional RNA-seq. Incorporation of the nucleoside analog 4-thiouridine into RNA allows the separation of nascent transcripts though reversible coupling to biotin and streptavdin bead pull-down1-4. The protocol takes roughly two days to generate a sequencing ready library.
- 105 cells in suspension
- TRIS, pH 7.5 (ThermoFisher: 15567027)
- EDTA (Invitrogen: AM9760G)
- DEPC-treated water (Ambion: AM9915G)
- NaCl, 5 M (Sigma: S9625)
- Tween 20 (Sigma: P9416)
- 4‐thiouridine, 4sU (Sigma: T4509) [500 mM in PCR grade water]
- PBS (Invitrogen: 10010031)
- TRI reagent (Sigma: T9424)
- RNAseZap (Sigma: R2020)
- Chloroform (Sigma: C2432)
- Glycoblue (ThermoFisher: AM9515)
- Isopropanol (Sigma: I9516)
- Ethanol (Sigma: 51976)
- MTSEA-biotin-XX (Biotium: #90066) [0.1 mg/mL in dimethylformamide]
- Dimethylformamide (Sigma: PHR1553)
- μMacs (Miltenyi: 130-074-101)
- Chloroform/isoamylalcohol, 24:1 (Sigma: 25668)
- Phase-lock tube (5Prime: 2900309)
- Dithiothreitol, DTT (Merck: 1114740025) [Make Fresh, 100 mM in PCR grade water]
- RNeasy RNA clean-up kit (Qiagen: 74204)
- QuantiFluor RNA kit (Promega: E3310)
Buffers:
10x Biotinylation buffer (25 µL per sample):
- 100mM Tris, pH 7.4
- 10mM EDTA
- DEPC-treated water
MACS wash buffer (4 mL per sample):
- 100mM Tris, pH 7.5
- 10mm EDTA
- 1M NaCl
- 0.1% Tween 20
- DEPC-treated water
- Nanodrop One, or quivalent (ThermoFischer Scientific: ND-ONEC-W)
- Qubit 4 Fluorometer, or quivalent (ThermoFischer Scientific: Q33238)
- MACS MultiStand (Miltenyi: 130-042-303)
- µMACS Separator (Miltenyi: 130-042-602)
4sU Incorperation
** Prepare 105 cells per mL of media **
1. Add 10 µL of 500 mM 4‐thiouridine (4sU) per mL of cells for a final concentration of 500 μM.
2. Incubate under standard growth conditions for 45 minutes (e.g. 37°C, 5% CO2)
3. Pellet the cells by centrifugation at 200 rcf for 5 min.
4. Discard supernatant and resuspend cells in an equal volume of PBS.
5. Pellet the cells by centrifugation at 200 rcf for 5 min.
6. Resuspend pellet in 1 mL of TRI reagent and transfer to microcentrifuge tube.
7. Snap freeze cells using liquid nitrogen OR dry ice and ethanol.
** Store lysed cells at -80ºC for up to 3 months **
RNA Extraction
** Chill centrifuge to 4ºC **
1. Thaw TRI reagent lysed cells on ice.
2. Add 200 μL chloroform.
3. Add 1 μL glycoblue and close tube securely.
4. Shake the tube vigorously for 15 sec.
5. Incubate at room temperaturefor for 2–3 min.
5. Centrifuge for 30 min at 12,000 rcf (4°C).
6. Transfer the upper aqueous phase to a new collection tube containing 500μL isopropanol.
7. Incubate for 10 min at room temperature.
8. Centrifuge for 10 min at 12,000 rcf (4°C).
9. Discard supernatant and wash the RNA pellet with with 700 µL 70% ethanol in DEPC-treated water.
10. Centrifuge for 10 min at 12,000 rcf (4°C).
11. Discard supernatant and air dry for 10 min.
12. Re-suspend in 20 µL DEPC-treated water.
13. Measure RNA concentration using a Nanodrop.
4sU Enrichment
** Pre-spin one phase-lock tube per sample **
** Heat 2 mL MACS wash buffer to 65°C **
1. Combine 20-100 μg of total RNA, 50 μL MTSEA-biotin-XX, 25 µL 10x Biotinylation buffer and sufficient DEPC-treated water to generate a 250 μL reaction.
2. Incubate for 30 min at room temperature with rotation.
3. Add 400 μL chloroform/isoamylalcohol (24:1).
4. Add 1 μL glycoblue.
5. Mix and incubate for 3 min.
6. Transfer mixture to a phase-lock tube.
7. Centrifuge for 5 min at 12,000 rcf (room temp).
8. Transfer upper phase (~230 µL) to a new tube.
9. Add 1:10 volume 5 M NaCl and an equal volume of isopropanol and mix.
10. Centrifuge for 20 min at 20,000 rcf (4ºC).
11. Discared supernatant and wash pellet with 500 µL of 75% ethanol in DEPC-treated water.
12. Centrifuge for 20 min at 20,000 rcf (4ºC).
13. Discared supernatant and resuspend pellet in 60μl DEPC-treated water.
14. Denature RNA at 65°C for 10 min followed by rapid cooling on ice for 5 min.
15. Incubate RNA with 60 μL streptavidin magnetic beads for 15 min at room temperature with rotation.
16. Place a µMACS column into a magnetic separator with a 15 mL waste collection tube.
17. Add the biotinylation reaction to the coulmn.
18. Wash the column with 1 mL 65ºC MACS was buffer, repeat for a total of two washes.
19. Wash the column with 1 mL room temp MACS was buffer, repeat for a total of two washes.
20. Replace the waste tube with a collection microcentrifuge tube.
21. Elute RNA from the column by adding 100 μL of freshly prepared 100 mM dithiothreitol (DTT), followed by a second elution with another 100 µL 5 min later.
22. Clean up labelled RNA using a Qiagen RNeasy RNA clean-up kit with on-column DNase digestion.
23. Elute purified RNA in 20 μL of DEPC-treated water.
24. Measure RNA concentration using the QuantiFluor RNA kit.
Further steps:
A. Index RNA using SMARTer Stranded Total RNA-Seq Kit v2 - Pico Input Mammalian following the manufacturer’s instructions with a fragmentation time of 3 min and 14 cycles of PCR amplification.
B. Sequence using 75-cycle paired end reads.
- To avoid RNA degradation thoroughly clean bench surfaces and pipettes with RNAseZAP.
- Cell centrifugation speeds are optimised for erythroid progenitor cells and may need adjusting.
1. Rabani, M. et al. Metabolic labeling of RNA uncovers principles of RNA production and degradation dynamics in mammalian cells. Nature biotechnology 29, 436-42. doi:10.1038/nbt.1861 (2011)
2. Hay, D. et al. Genetic dissection of the α-globin super-enhancer in vivo. Nature genetics vol. 48, 895-903. doi:10.1038/ng.3605 (2016)
3. Friedel, C.C. et al. Conserved principles of mammalian transcriptional regulation revealed by RNA half-life. Nucleic acids research 37, e115. doi:10.1093/nar/gkp542 (2009)
4. Duffy, E.E et al. Tracking Distinct RNA Populations Using Efficient and Reversible Covalent Chemistry. Molecular cell 59, 858-66. doi:10.1016/j.molcel.2015.07.023 (2015)
This protocol has been posted on Protocol Exchange, an open repository of community-contributed protocols sponsored by Nature Portfolio. These protocols are posted directly on the Protocol Exchange by authors and are made freely available to the scientific community for use and comment.
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