** 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 **
** 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.
** 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.
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.