I feel pretty silly asking this, but am stumped.

I'm attempting to do dCas9-CAPTURE (paper: 10.1016/j.cell.2017.08.003) and am having issues during the chromatin isolation step - the chromatin does not pellet. Each time I've tried this I've prepared the buffers fresh. The chromatin appears to precipitate, but does not pellet on the spin step (step g in the protocol below). The methods in the linked paper are somewhat terse. I'm attempting to follow the bit with the subheading "dCas9 affinity purification". My adaption of their methods is outlined below:

a. count cells (previously fixed and frozen)

b. resuspend in 10 mL of CAPTURE cell lysis buffer (25 mM Tris-HCl, 85 mM KCl, 0.1% Triton X-100, pH 7.4, freshly added 1 mM DTT and 1:200 PIC) I have this buffer already made up. I will pH a bit of it to check that it hasn't gone off.

c.rotate 15 minutes at 4°C

d.centrifuge at 2300xg for 5 minutes at 4°C to isolate nuclei.

e.resuspend nuclei in 5ml CAPTURE nuclear lysis buffer (50 mM Tris-HCl, 10 mM EDTA, 4% SDS, pH 7.4, freshly added 1 mM DTT and 1:200 PIC)

f.incubate for 10 minutes at room temperature on rotator

g.mix with 3 volumes (15ml) 8M urea and centrifuge at 16,100 x g for 25 min at room temperature.

h.aspirate super.

i.wash 3x by resuspending in 5ml nuclear lysis buffer, adding 15ml 8M urea, and spinning 16,100 x g for 25 min at room temp.

j.wash 2x with 5ml CAPTURE cell lysis buffer.

k.Resuspend in 5 mL of IP binding buffer without NaCl (20 mM Tris-HCl, 1 mM EDTA, 0.1% NP-40, 10% glycerol, pH 7.5, freshly added PIC).

[...]

Because I can see the precipitated chromatin in solution, but it appears not to pellet, I figured that the issue may be the density of lysis buffer-urea solution? I am unsure. How much could the density change with temperature? Is it possible that the difference in temperature in their "room temp" centrifuge and mine is enough to make the solution more dense and hence make the chromatin positively buoyant? When you read room-temp centrifugation, do you assume a 25 minute spin with no refrigeration, or a 25 minute spin with the centrifuge set to 20°C? Is there some other glaring problem or omitted step you see that I am missing, or that is missing from the methods of the linked paper?