Protocol Version: 10.27
Date in original DOC: 2020.11.12
Author: Amy Pandya-Jones
Adapted from: Wuarin J & Schibler U, MCB, 1994

This protocol is for 5–10 × 10⁶ cells or one confluent 10 cm plate.

Important Notes

Make all solutions the day before to allow the detergent sufficient time to dissolve.
Recommend making a large volume of each buffer (e.g., 100 mL) to maintain consistency.
Filter sterilize all buffers (sucrose buffer is easily contaminated).
This protocol is optimized for RNA isolation but can be used for protein too.
For protein isolation: add protease/phosphatase inhibitors to the buffers (fresh).
From the cell pelleting step onward: KEEP CELLS ON ICE AND BUFFERS COLD.
Do not pipette cells to resuspend — gently flick the tubes instead.
For chromatin protein westerns: do not centrifuge the chromatin protein sample before loading; chromatin/histones pellet easily and the supernatant will be histone-poor.

Procedure

1. Cell Harvesting

Trypsinize cells from plate.
Collect cells by adding 10 mL media and count cell number.
Pellet by low speed centrifugation (500 g, 5 min, room temperature, 15 mL falcon tube).
Aspirate media.
Gently re-suspend cell pellets in 1 mL PBS/1 mM EDTA and transfer to 1.7 mL Eppendorf tube.
Pellet cells again by low speed centrifugation (~500 × g in an Eppendorf 5415C centrifuge at RT).

2. Cytoplasmic Fraction Extraction

Add 200 μL ice-cold NP-40 lysis buffer to the cell pellet.

NP-40 Lysis Buffer composition:
10 mM Tris-HCl (pH 7.5), 0.05–0.15% NP-40, 150 mM NaCl

Flick to resuspend.
Incubate on ice for 5 minutes.

Cell type optimization (keep lysis time constant; adjust NP-40 concentration or buffer volume instead):

Cell Type	Cell Number	Lysis Buffer Vol	NP-40 conc.	Incubate time
NCCIT	6 million	200 μL	0.15%	5 min

Guidelines:

Neuronal cells: 0.05% NP-40
HEK293 and ES cells: 0.1% NP-40
HeLa cells: 0.15% NP-40

Gently pipette up the cell lysate and layer over 2.5 volumes (500 μL) of chilled sucrose cushion (24% RNase-free sucrose in lysis buffer).
Centrifuge for 10 min, 4°C, 15000 × g in an Eppendorf 5415C centrifuge.

Note: Nuclei can lyse if centrifugal speed is too high — calculate RCF carefully.

3. Cytoplasmic Fraction Collection

Collect all ~500 μL of supernatant (cytoplasmic fraction).
Take 100 μL (1/10) aliquot for monitoring fractionation by western.
Additional 100 μL supernatant together with Soluble nucleoplasmic fraction can be used for RNA extraction.
To the remaining cytoplasmic extract:

Add equal volume of 2× proteinase K buffer
Add proteinase K (20 μL of 20 mg/mL)
Incubate at 37°C for 1 h
Phenol/chloroform extract and ethanol precipitate RNA
Alternative: Use Qiagen RNeasy kit (lower yields)

4. Soluble Nucleoplasmic Fraction Extraction

Gently add ~200 μL ice-cold PBS/1 mM EDTA to the nuclei pellet.
Aspirate the PBS/EDTA (this rinses away leftover cytoplasmic extract).

Nuclei should appear as a light white cloud.
Yellowish parts indicate unlysed cells or released chromatin.

Resuspend nuclei pellet in 100 μL prechilled glycerol buffer by gentle flicking.

Yellowish chromatin clumps won’t resuspend.
Intact nuclei give a homogeneous, slightly opaque solution.

Add equal volume (100 μL) of cold nuclei lysis buffer.
Vortex vigorously (highest setting) for 2 × 2 seconds.
Incubate for 2 min on ice.
Centrifuge for 2 min, 4°C, 15000 × g.

Note: The 2-minute incubation with high NP-40 and urea strips loosely associating soluble material from chromatin. The RNA-Pol II–DNA ternary complex is resistant to these conditions.

5. Soluble Nucleoplasmic Fraction Collection

Collect supernatant (soluble nuclear fraction / nucleoplasm).
Take 20 μL (1/10) aliquot for western analysis.
For RNA isolation: treat with proteinase K or use Qiagen RNeasy column.

6. Chromatin-associated Fraction

Gently rinse pellet with cold PBS/1 mM EDTA (as done for nuclei pellet).
Treat pellet (in 100 μL) with RNase-free DNase (Roche, 20 U) for 5–10 min at 37°C.

Take 10 μL (1/10) aliquot for western analysis.
This fraction will be almost devoid of histones if spun down; do not spin chromatin sample before western.

Dissolve remaining chromatin (~50 μL) in 1 mL TRIzol (Invitrogen).

This can be difficult due to high DNA content.
Place in a 50°C heatblock until DNA dissolves.
Perform an additional phenol/chloroform extraction step after aqueous fraction isolation.

RNA Extraction Using Phase-Lock Tubes

Spin down phase-lock tubes as instructed (16000 g, 30 s).
Add 200 μL samples to each tube.
Add 1 volume (200 μL) 25:24:1 Phenol–Chloroform–Isoamyl alcohol.
Mix by fierce shaking for at least 50 times.
Spin 5 min at 13200 rpm (max) at 16°C.
Transfer aqueous phase to a new phase-lock tube.
Add 300 μL chloroform, shake vigorously, spin again (same conditions).
Transfer final aqueous layer to a fresh low-binding tube.
Add:

1/10 volume 3 M NaOAc
1 μL glycogen (carrier)
3 volumes 100% ethanol

Mix and precipitate overnight at −80°C.
Next day, spin down RNA pellet, wash with 70% ethanol, air-dry briefly, and resuspend in RNase-free water.

Buffer Recipes

1× PBS / 1 mM EDTA (50 mL)

Add 0.5 mL 0.5 M EDTA to 50 mL PBS.

NP-40 Lysis Buffer (2× 50 mL)

Reagent	Stock	Volume (50 mL)	Final Concentration
Tris-HCl (pH 7.5)	1 M	0.5 mL	10 mM
NP-40 (Sigma)	10%	0.5 mL	0.05–0.15%
NaCl	5 M	1.5 mL	150 mM
ddH₂O	—	47.5 mL	—

Sucrose Cushion (24% RNase-free sucrose in lysis buffer)

Dissolve 12 g sucrose in lysis buffer and bring to 50 mL.

Glycerol Buffer (50 mL)

Reagent	Stock	Volume	Final Concentration
Tris-HCl (pH 7.9)	1 M	1 mL	20 mM
NaCl	5 M	0.75 mL	75 mM
EDTA	0.5 M	50 μL	0.5 mM
DTT	1 M	42.5 μL	0.85 mM
PMSF	100 mM	62.5 μL	0.125 mM (add fresh)
Glycerol	100%	25 mL	50%
ddH₂O	—	to 50 mL	—

Add 1× protease inhibitor cocktail fresh before use if isolating proteins.

Nuclei Lysis Buffer (50 mL)

Reagent	Stock	Volume	Final Concentration
HEPES (pH 7.6)	1 M	0.5 mL	10 mM
DTT	1 M	50 μL	1 mM
MgCl₂	1 M	0.375 mL	7.5 mM
EDTA	0.5 M	20 μL	0.2 mM
NaCl	5 M	3 mL	0.3 M
Urea	8 M	6.25 mL	1 M
NP-40	10%	5 mL	1%
ddH₂O	—	to 50 mL	—

8 M urea stock: dissolve 2.4 g urea in ddH₂O and bring to 50 mL.

Add 1× protease inhibitor cocktail fresh before use if isolating proteins.

DNase Reaction Buffer (for chromatin step)

(Prepare according to enzyme manufacturer; keep RNase-free.)
Typical final conditions include Mg²⁺-containing buffer suitable for RNase-free DNase I.

Final Sample Table (record your conditions)

Sample	Cell Type	Cell #	NP-40 (%)	Lysis Vol (μL)	Cytoplasm Yield Notes
1
2
3

References

Wuarin J & Schibler U, Molecular and Cellular Biology, 1994
Cai H & Luse DS, Journal of Biological Chemistry, 1987

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