Cell lysis

Are there protocols for nuclear vs cytoplasmic CLIP for shuttling RBPs?

Several publications, including from Ule lab, have done this. I add the relevant part from an old protocol below, this is meant to be a quick fractionation to minimise RNase activity, it may need to be optimised for your types of cells/tissues:

Cytoplasmic Lysis buffer (CLB):

• 50 mM Tris-HCl, pH 7.4

• 10mM NaCl

• 0.5% Igepal CA-630 (Sigma I8896)

• 0.25% Triton X-100

• 1mM EDTA

On the day of the experiment, add complete protease inhibitor cocktail (Roche, 25x) to the amount of buffer required for lysis.

CBP-adjusting buffer:

• 50 mM Tris-HCl, pH 7.4

• 100 mM NaCl

• 4% Igepal CA-630 (Sigma I8896)

• 0.4% SDS

• 2% sodium deoxycholate

Nucleus Lysis Buffer (NLB)

• 50 mM Tris-HCl, pH 7.4

• 100 mM NaCl

• 1% Igepal CA-630 (Sigma I8896)

• 0.1% SDS

• 0.5% sodium deoxycholate

On the day of the experiment, add cOmplete protease inhibitor cocktail (Roche, 25x) to the amount of buffer required for lysis.

2.3 A. Resuspend cell pellet (from step 1, each pellet is usually enough for 1 IP) in 1.5 ml of cold CLB. Make sure to fully resuspend the pellet by pipetting up and down with a 1ml pipette tip, and then rotate in cold room for 5 minutes. Afterwards spin at 4C, 1000 x g for 3 minutes. Collect the supernatant for cytoplasmic iCLIP and add 0.5 ml of CBP-adjusting buffer.

2.3 B. Resuspend the pellet in 1ml CLB, transfer to a 1.5 mL tube and spin again at 4C, 1000 x g for 3 minutes. Discard the supernatant, and add 1ml of NLB to the pellet (supplemented with protease inhibitors). Then use Bioruptor for 10 cycles with alternating 30 secs on/ off at low intensity. Six samples can be sonicated at the same time.

2.3 C. Measure lysate concentration with Bradford assay and dilute all samples with NLB to 1 mg/ml.

RNase

We were not able to get a clean result after end-labeling of the RNA (Step-8) like you showed in Figure 2. In our trials, lane 1 and 2 (no antibody controls) were blank, just like what you showed in the figure. But our lane 3 and 4 both have a smear in the entire lane (even below the size of our protein), and the signal in lane 3 (the high RNase treated sample) was only slightly lower than lane 4 (low RNase). There was a band in lane 3 (high RNase) but not lane 4 (low RNase) at the size of our protein, but it was accompanied by significant amount of smear. Do you know why there was so much signal below the size of our protein, and how we can get rid of the smear in the high RNase treated sample?

This is a phenomenon that also occurred to us in the past. Most often, it results from immunoprecipitation conditions that are not stringent enough for the IPed protein, which allow co-purification of other associated RNA-binding proteins that migrate below the size of the protein. For this purpose, it would help to try a few different IP conditions - sometimes it is sufficient just to increase the volume of lysis buffer added to the pellets and incubate the beads 5min rotating in cold room in the high-salt wash buffer during washes. An alternative is to increase the concentration of RNAse in the high-RNAse condition. It’s likely that the concentration isn’t high enough for your conditions.

RNase inhibitors

How do RNasin (Promega) and anti-RNase (Ambion) compare?

They are similar RNAses, we use them in the lysis buffer when we wish to selectively inhibit RNase A, but not RNase I. Otherwise, SUPERaseIn (Life Technologies, AM2696) can be used if you want to also inhibit RNase I.

Sonication & getting rid of the DNA

After RNase/Turbo DNase treatment of the lysate and centrifugation of the lysate, intact chromosomes were not completely precipitated and took up most of lysate volume as viscous goo. I could not pipet out the sup since pipet tips caught the goo very easily. I wonder Turbo DNase did not work well enough. A short sonication would help to chop off DNAs, but it would also shear RNAs. Would it make sense to increase Turbo DNase (amount and/or length), or to pretreat the lysate with Turbo DNase for some time before adding RNase? I could spin the pellet with higher than 20,000xG by using ultracentrifuge if it is an easier solution.

Sonication is the only solution that works for us. Shearing of RNA is not a problem, since in any case we want to partially digest the RNA.