Table 1.

Troubleshooting Advice

STEP	PROBLEM	POSSIBLE REASON	SOLUTION
7A(iii) & 7B(iii)	Nuclei do not pellet well	Cells are not completely lysed	Incubate on ice longer and use more vigorous pipetting to resuspend cells in Step 7A(ii) or 7B(ii).
		Nuclei are smaller than typical	Centrifuge at higher speed or for longer duration.
		Washes are incomplete	Wash additional times or with more vigorous pipetting during resuspension.
7B(iv)	Nuclei are not completely lysed	Sonication ineffective	Increase sonication time or sonicator power output.
7B(iv)	Nuclear extract is foamy	Oversonication	Decrease sonication time or sonicator power output.
7B(iv)	Nuclear extract is viscous	genomic DNA not sufficiently sheared	Increase sonication time or sonicator power output.
Box 1	Nuclei are not oval or rounded	nuclear membrane damaged	wash nuclei with fewer washes or gentler pipetting in Step 7A(ii) or 7B(ii).
Box 1	DAPI staining outside of nuclei	nuclear membrane damaged	wash nuclei with fewer washes or pipette more gently in Step 7A(ii) or 7B(ii).
			check to ensure NP-40 concentrations are correct during Reagent Setup.
Box 1	Signal from ER tracker dye	ER membrane proteins not efficiently removed	increase wash times, NP-40 concentration, or pipetting stringency in Step 7A(ii) or 7B(ii).
		tracker dye concentration too high	check tracker dye concentration, ensure nuclei are diluted after incubation.
Box 1	No ER tracker signal from controls	ER tracker dye concentration too low	increase tracker dye concentration incubation or time.
Box 2	RNA radioactivity is low	Inefficient labeling	Increase [λ]-32P-ATP, PNK enzyme, or reaction duration in Step (i) of Box 2.
		Inefficient gel extraction	Use more water during elution from gel in Step (viii) of Box 2.
			Recover gel bits off of tip by pipetting buffer down the tip Step (viii) of Box 2.
			Add more tRNA and LiClO4 in acetone to RNA solution during precipitation in Step (x) of Box 2.
10A(v) & 10B(v)	Bands are not visible	Inefficient siRNA loading	Add higher amounts of radioactive RNA to the loading reaction in Step 9A(vi) or 9B(i).
			Increase extract volume or use more antibody and resin in Step 9A(ii) or 9B(ii–iv).
			Incubate loading reactions longer in Step 9A(vi) or 9B(i) & 9B(iv).
		Inefficient Ago2 immunoprecipitation	Increase amount of antibody or resin during immunoprecipitation in Step 9A(ii) or 9B(ii–iv).
			Incubate antibody and resin with extract longer in Step 9A(ii) or 9B(iii–iv).
			Reduce wash duration or wash buffer NaCl concentration in Step 9A(vii) or 9B(v).
		Extract quality poor	Optimize fractionation in Step 7B.
		Ago2 expression low	Increase extract volume during loading reactions in Step 9A(ii) and 9B(i).
10A(v) & 10B(v)	Bands are blurry or heterogeneous	Poor gel quality	Optimize gel pouring conditions in Step 10A(i) and 10B(i) or purchase commercial gels.
		Gel ran too hot or too fast	Reduce current or improve cooling in Step 10A(ii–iii).
		Gel ran too cool or too slow	Increase current in Step 10B(ii–iii).
		Gel ran too long	Reduce run time in Step 10A(ii) or 10B(ii).
10A(v) & 10B(v)	Bands are visible in IgG controls	Inefficient resin washing	Wash longer, wash with more buffer, wash more times or wash with a higher NaCl concentration in the wash buffer in Step 9A(vii) or 9B(v).
		Overexposed phosphorimager screen	Reduce exposure time to phosporimager screen in Step 10A(v) or 10B(v).