2.2 Isolation of
Nuclei
1. Chopping Buffer: 45 mM MgCl 2 , 30 mM sodium citrate,
20 mM 3-(N-morpholino) propane sulfonic acid (MOPS),
pH 7.0 adjusted with NaOH. Filter sterilize and store as
50 mL aliquots at -20
C. Once thawed, buffer may be kept
at 4
C for up to 1 week.
2. Propidium iodide (PI) stock solution: 1 mg/mL prepared in
deionized water (diH 2 O). Store as 1 mL aliquots at –20
C
until the day of use.
3. 10 mg/mL Solution of DNAse-free RNAse A prepared in
diH 2 O. Store as 100 μL aliquots at –20
C.
4. 4
0 ,6-Diamidino-2-phenylindole (DAPI) stock solution:
0.1 mg/mL prepared in diH 2 O. Store as 1 mL aliquots at –
20
C until the day of use.
5. 10 μm Flow-Check™ Fluorospheres (Beckman Coulter, Fullerton, CA).
6. Supplies: Double-edged shaving razor blades; plastic razorblade holders; Sysmex CellTrics
® 30 μm pore size disposable
mesh filters.
2.3 Preparing Nuclei
for 10Â Genomics
Processing
1. RNase-free Sheath Buffer: 0.01 M PBS or 0.9% NaCl, prepared
just before the day of use.
0.01 M PBS: 22.4 g PBS dissolved in 2 L deionized water
(diH 2 O), adjusted to pH 7.2–7.4. Add 2 mL diethylpyrocarbonate (DEPC) and vortex for 10 hours. Filter through a
0.44 μm filter membrane and autoclave.
0.9% NaCl: 18 g NaCl dissolved in 2 L diH 2 O and adjust
pH to 7.2–7.4. Add 2 mL DEPC and vortex for 10 h. Filter
through a 0.44 μm filter membrane and autoclave.
2. RNase-free Galbraith Buffer: 45 mM MgCl 2 , 30 mM sodium
citrate, and 20 mM MOPS in DEPC-treated diH 2 O, pH 7.0
adjusted with NaOH. Filter sterilize and store as 50 mL aliquots at À20
C. Once thawed, buffer may be kept at 4
C for
up to 1 week.
3. BSA stock solution: 2% BSA prepared in DEPC-treated
diH 2 O. Filter sterilize and store at À20
C until the day of use.
4. Propidium iodide (PI) stock solution: 1 mg/mL prepared in
DEPC-treated diH 2 O. Store as 1 mL aliquots at À20
C until
the day of use.
5. DTT stock solution: 1 M DTT in DEPC-treated diH 2 O. Store
as 1 mL aliquots at À20
C until the day of use.
6. Recombinant RNasin
® ribonuclease inhibitors: 2500 U (Promega, 40 U/μL stock solution), store at À20
C until the day
of use.
7. RNAse inhibitor, 2500 U (40 U/μL, Invitrogen). Store at
À20
C until the day of use.
Flow Cytometry and Sorting in Arabidopsis
271
Nuclei
1. Chopping Buffer: 45 mM MgCl 2 , 30 mM sodium citrate,
20 mM 3-(N-morpholino) propane sulfonic acid (MOPS),
pH 7.0 adjusted with NaOH. Filter sterilize and store as
50 mL aliquots at -20
C. Once thawed, buffer may be kept
at 4
C for up to 1 week.
2. Propidium iodide (PI) stock solution: 1 mg/mL prepared in
deionized water (diH 2 O). Store as 1 mL aliquots at –20
C
until the day of use.
3. 10 mg/mL Solution of DNAse-free RNAse A prepared in
diH 2 O. Store as 100 μL aliquots at –20
C.
4. 4
0 ,6-Diamidino-2-phenylindole (DAPI) stock solution:
0.1 mg/mL prepared in diH 2 O. Store as 1 mL aliquots at –
20
C until the day of use.
5. 10 μm Flow-Check™ Fluorospheres (Beckman Coulter, Fullerton, CA).
6. Supplies: Double-edged shaving razor blades; plastic razorblade holders; Sysmex CellTrics
® 30 μm pore size disposable
mesh filters.
2.3 Preparing Nuclei
for 10Â Genomics
Processing
1. RNase-free Sheath Buffer: 0.01 M PBS or 0.9% NaCl, prepared
just before the day of use.
0.01 M PBS: 22.4 g PBS dissolved in 2 L deionized water
(diH 2 O), adjusted to pH 7.2–7.4. Add 2 mL diethylpyrocarbonate (DEPC) and vortex for 10 hours. Filter through a
0.44 μm filter membrane and autoclave.
0.9% NaCl: 18 g NaCl dissolved in 2 L diH 2 O and adjust
pH to 7.2–7.4. Add 2 mL DEPC and vortex for 10 h. Filter
through a 0.44 μm filter membrane and autoclave.
2. RNase-free Galbraith Buffer: 45 mM MgCl 2 , 30 mM sodium
citrate, and 20 mM MOPS in DEPC-treated diH 2 O, pH 7.0
adjusted with NaOH. Filter sterilize and store as 50 mL aliquots at À20
C. Once thawed, buffer may be kept at 4
C for
up to 1 week.
3. BSA stock solution: 2% BSA prepared in DEPC-treated
diH 2 O. Filter sterilize and store at À20
C until the day of use.
4. Propidium iodide (PI) stock solution: 1 mg/mL prepared in
DEPC-treated diH 2 O. Store as 1 mL aliquots at À20
C until
the day of use.
5. DTT stock solution: 1 M DTT in DEPC-treated diH 2 O. Store
as 1 mL aliquots at À20
C until the day of use.
6. Recombinant RNasin
® ribonuclease inhibitors: 2500 U (Promega, 40 U/μL stock solution), store at À20
C until the day
of use.
7. RNAse inhibitor, 2500 U (40 U/μL, Invitrogen). Store at
À20
C until the day of use.
Flow Cytometry and Sorting in Arabidopsis
271