TITLE: Isolating enriched fractions of nuclear and cytoplasmic RNAs from Arabidopsis flowers
AUTHORS: Diep R Ganguly, Wil Prall, Susheel Sagar Bhat, Sean Hilgendorf, Brian D Gregory

[DESCRIPTION]
This is an adapted protocol to isolate RNA fractions enriched for nuclear and cytosolic RNAs from frozen Arabidopsis flowers using the Norgen Biotek Cytoplasmic and Nuclear RNA Purification Kit (Product # 21000) alongside the RNase-Free DNase I Kit (Norgen, # 25710).

[BEFORE_START]
- Prepare an RNase-free environment (e.g. clean bench and pipettes, filter tips, nuclease-free solutions).
- Make sure plant material has been frozen and ground into a fine powder.
- Prepare 200 μL/sample Lysis Buffer J containing 10 μL/mL 2-mercaptoethanol (prepare before use).
- Prepare 600 μL/sample Buffer SK containing 10 μL/mL 2-mercaptoethanol (prepare before use).
- Ensure Wash Solution A contains ethanol.
- Ensure you have working aliquots of RNase-free DNase I (Norgen).

[GUIDELINES]
- 20-50 mg of Arabidopsis flowers yields approximately 20 μg cytosolic- and 5 μg nuclear-enriched RNA.
- Perform qRT-PCR on the resulting fractions with primers targeting mature mRNAs and pre-mRNAs (i.e. primers binding intron-exon junctions) to quantify enrichment. For example, we quantify pre-mRNA (nuclear-enriched) and mature mRNA (cytosol-enriched) of PP2AA3 (AT1G13320, mRNA: F - CGACCAAGCGGTTGTGGAGA, R -  CACAATTCGTTGCTGTCTTCTTT; pre-mRNA: F - GCTTTATGGGAAAGCTGTAAGG, R - CAACCATATAACGCACACGCC).
- We also quantify long non-coding RNAs lncCOBRA3 (AT3G03435, F - GTTGAGTCGCTTCGTCTATGT, R - TGCCATCATAGGATCCTTCAATAA) and lncCOBRA5 (AT3G05655, F - TGCACAAGTACTGGGACATC, R - GATTCGGGTCGGGTCATAAG), which show a strong enrichment in the nucleus (Kramer et al 2022, Frontiers in Plant Science).

[STEPS]
SECTION: Lyse cells and fraction RNA
1. Grind frozen Arabidopsis flowers into a fine powder using a pestle. Keep samples frozen in liquid nitrogen until you are ready to add lysis buffer.
SECTION: Lyse cells and fraction RNA
2. Remove samples from liquid nitrogen and immediately add Lysis Buffer J (200 μL per 20-50 mg tissue).
SECTION: Lyse cells and fraction RNA
6. Transfer supernatant (cytoplasmic RNA) to a clean nuclease-free tube.  Be careful not to disturb the pellet (nuclear RNA).
10. Add 200 μL of 100% ethanol to all samples then mix vigorously for 10 seconds.
12. Discard flow through and reassemble spin column on collection tube.
SECTION: Bind RNA onto column
7. Add 200 μL Buffer SK to cytoplasmic RNA.
SECTION: Washing RNA and on-column DNase treatment (nuclear RNA only)
13. Add 400 μL of Wash Solution A to the column and centrifuge 2 minutes at 14,000 rcf.
SECTION: Washing RNA and on-column DNase treatment (nuclear RNA only)
15. 
For every on-column nuclear RNA sample, mix 15 μL DNase I with 100 μL enzyme incubation buffer from the RNase-free DNase I kit (Norgen Biotek, # 25710). Mix by gentle inversion.
SECTION: Washing RNA and on-column DNase treatment (nuclear RNA only)
16. Add 100 μL of RNase-free DNase I to columns with nuclear RNA and centrifuge for 1 minute at 18,000 rcf (ensure entire DNase I solution passes through filter). 
SECTION: Washing RNA and on-column DNase treatment (nuclear RNA only)
17. Pipette flow through back onto column and incubate at 25 - 30 °C for 15 minutes.
19. Perform a final wash to all tubes, discard flow through, and reassemble spin column on collection tube.
20. Spin the column for 2 minutes at 18,000 rcf to thoroughly dry the resin.
SECTION: Elution
21. Transfer column to clean nuclease-free tube (discard the collection tube).
SECTION: Elution
22. Add 50 μL of Elution Buffer E (or 10 mM Tris-Cl, 0.1 mM EDTA, pH 6.5).
SECTION: Elution
23. Centrifuge for 2 minutes at 200 rcf, then 1 minute at 18,000 rcf. 
11. Transfer lysate to a spin column on a collection tube then centrifuge for 1 minute at 18,000 rcf.
SECTION: Washing RNA and on-column DNase treatment (nuclear RNA only)
14. Discard flow through and reassemble spin column with collection tube.
18. Add 400 μL of Wash Solution A to all columns, centrifuge for 1 minute at 18,000 rcf, then discard flow through.
SECTION: Elution
24. Perform a second elution step for maximum RNA recovery.
SECTION: Ethanol precipitation (optional)
25. Add 0.1x volume NaOAc (3 M, pH 5.5) to samples.
SECTION: Ethanol precipitation (optional)
26. Add Glycogen or GlycoBlue to samples to a final concentration of 50 μg/mL.
SECTION: Ethanol precipitation (optional)
30. Incubate samples for at least 1 hour at -20 °C (O/N to maximize recovery).
SECTION: Ethanol precipitation (optional)
31. Centrifuge at max speed for 30 min at 4 °C.
SECTION: Ethanol precipitation (optional)
32. Remove supernatant and rinse pellet with 750 μL 70% (v/v) ethanol.
SECTION: Ethanol precipitation (optional)
33. Centrifuge at 9,200 rcf for 10 min at 4 °C.
SECTION: Ethanol precipitation (optional)
34. Remove supernatant without disturbing pellet and allow to air dry for 1-2 min.
SECTION: Ethanol precipitation (optional)
35. Resuspend in desired volume of nuclease-free water or Tris-EDTA (10 mM Tris-Cl, 0.1 mM EDTA, pH 6.5).
SECTION: Quality control
37. Assess quantity and quality of RNA (e.g. visualize heat-treated RNA on a 1% TBE agarose gel, run on bioanalyzer or LabChip, Nanodrop, and/or Qubit).
SECTION: Quality control
38. Perform qRT-PCR to test for level of enrichment by, for example, comparing mature mRNA and pre-mRNA levels in each fraction (using primers spanning intron-exon junctions).
SECTION: Lyse cells and fraction RNA
5. Centrifuge lysate for 10 minutes at max speed.
SECTION: Ethanol precipitation (optional)
28. Add 3-5x volumes of 100% ethanol to samples.
SECTION: Lyse cells and fraction RNA
3. Mix samples with gentle inversion until tissue is completely dissolved.
SECTION: Ethanol precipitation (optional)
29. Mix by gentle inversion.
SECTION: Lyse cells and fraction RNA
4. Incubate for 3 minutes at ambient temperature.
SECTION: Bind RNA onto column
8. Add 400 μL Buffer SK to nuclear RNA.
SECTION: Bind RNA onto column
9. Mix all samples vigorously for 10 seconds.
SECTION: Ethanol precipitation (optional)
27. Mix by flicking tube.
SECTION: Ethanol precipitation (optional)
36. Store RNA at -20 ºC and -80 ºC for short and long term storage, respectively.