At the beginning of step 10 (and 11-13), after spinning for 20 mins, the only pellet I saw was a small blue fleck. this stayed in tact through the EtOH wash and then dissolved in water. Is there more of a pellet at the bottom of the tube that I just can't see or is this blue fleck the only thing I have to worry about not removing? I'm afraid there was RNA/cDNA at the bottom of my tube that I pipetted out in my attempt to remove all the alcohol (while being careful to leave the small blue fleck in the tube).
The blue fleck contains the RNA - so it’s the only thing to worry about ;-)
When the RNA or cDNA was precipitated and washed after extraction, some samples had relatively large pellets, which might not be completely soluble in the small amount of buffer (7.25uL) used to dissolve the pellet prior to RT. Obviously it was not made of pure RNA. I would think the pellets are mostly made up with salt or some component of nitrocellulose membrane. Would it be OK to just ignore it and go ahead?
Yes, the additional precipitate is most likely salt or other contamination. This may inhibit other biochemical steps, so I advice against proceeding. See Huppertz et al, Method 2014 manuscript, which discusses ways how to ensure that precipitations are clean.
We're trying to troubleshoot iCLIP experiments following your Jove protocol... what ladder did you use in Figure 3? We've had some issues where the amplified library size isn't what we expect given the size we're selecting for at the cDNA (pre-circular ligase) step, and we're trying to figure out what the cause is.
I agree this can be a problem of size selection, because linear cDNA runs more similar to the size of single-stranded RNA than double-stranded DNA marker, which are normally used. Therefore we suggest to load 6 µl RNA century size marker (Invitrogen AM7140; diluted 1/30, and stored as aliquots at -20) for the cDNA gel.
After reverse transcription, cDNA products are resolved in TBE-urea gels and a DNA low molecular weight marker is used (double strand!). Does this marker run correctly? As single strand? Or as a mixture of complementary bands?.
If denatured correctly this marker runs as single strands. Denaturing time can be prolonged to 5 min to ensure full denaturation. And don't use too much marker, because when overloaded it doesn't denature fully.
How did you see the ladder in 11.4?
You cut off the part of the gel that contains the ladder and stain it with Sybr green II.
During purification of the cDNA from the gel, you separately excise the "Low", Medium" and "High" RNA fractions. Do you sequence them separately as well? If yes, how do you barcode them (i.e. different barcodes for each fractions or the same barcodes for the same fraction from a different biological replica?) If you pool them together, at what stage do you do it?
We keep it separate during PCR. We don’t barcode them, but we do mix them after PCR if they all look good. Sometimes the shortest cDNAs contain primer dimmer - in this case we don’t sequence them. We can’t figure out after sequencing which sequence came from which band.