L3 linker DNA does not have a 3'-puromycin
It has ddC (dideoxycytosine), which works equally well to block ligation.
Why does the RT primer contain only nine "linker L3 specific" bases for the reverse transcription step and not the maximum (20)? If we don't have the barcode in the RT primer, do you think we should add twenty "linker L3 specific" bases for the reverse transcription instead of nine?
It's important that the RT primer doesn’t have all 20 of the L3-specific bases, otherwise circularised RT primer could serve as a template for PCR after linearization. Even though we run a cDNA gel after RT, the concentration of primer is so high that it can contaminate the purified cDNAs. We have made great effort in the early stages of iCLIP development, tried RT primers with different numbers of L3-specific bases, and found that having nine bases gave best results.
Do you somehow test the RT primers with different barcodes to ensure that they all have efficient priming?
We tested the oligos by splitting a single experiment before RT, and then comparing PCR products at the end. This can performed whenever ordering a new batch of primers. We initially found some variations between primer qualities when ordering non-purified DNA, and this variation was not related to their sequence (it depended on the synthesis batch, and might have to do with residual salt). However, now we order them as HPLC purified, and we don’t find much variation anymore.
All Rclip 1-16 primers have two consecutive wobbles (NN) at the 5'end immediately followed by a 4bp bar code and another 3bp-long wobble (NNN). What is the purpose of this?
The wobbles (NNN) are part of unique molecular identifier (UMI), also referred to as random barcode. Their analysis allows to distinguish individual cDNAs from PCR amplicons (see chapter ‘Use of random barcode in data analysis’). The three wobbles will be positioned at the start of sequencing read, which ensures efficient cluster identification by the Illumina software, which uses the first 4 cycles, and thus high sequence diversity is required in the first nucleotides. The two more wobbles at the 5'-end of the primer are added to avoid potential effects of defined barcodes on the efficiency of cDNA circularization.
We order the primer from IDT, and specify ‘N’ at the corresponding positions in the primer to define these wobbles.
The cut-oligo and Rclip RT primers do not have 5'-amino (6 carbon) group ( you mentioned that they are important in a Methods, CLIP paper, 2009).
They were important to block ligation of 5’ end of adapter at the time, but here the protocol is now different, and we do not need to block ligation.
We have recently had a problem with using rt5clip. In two independent experiments, rt5clip samples were taking over 90% of the reads, while the libraries using rt5clip were not necessarily looking different from the other libraries. I also noticed that rt5clip sequence was dropped from the Huppertz et al. 2014 Methods paper. What is the reason for this, did you observe a similar problem?
Yes, I believe we avoided rt5clip at the time, because we tested each ordered primer and saw this one being an outlier. That said, we interpreted it as variation in quality of synthesis rather than primer sequence, as no primer would reproducibly be an outlier across stocks. In your case, it seems that it is best to exclude your current rt5clip primer stock from future experiments too. Thank you so much for your fast answer, this answers all our worries!
What is the purpose of the cut oligo?
The cut oligo was used in the original variant of iCLIP, and was used to cut the linearized cDNA with BamH1.
Why does the Cut Primer contain "aaaa" at the end ?
To prevent it from being a template for PCR
What sort of primer I should use if I want to start by cloning my insert into TOPO vector instead of doing nextGen sequencing?
Same primer can be used as described in the protocol. TOPO cloning doesn’t require any specific primer.
Do P5 and P3 solexa primers bind the library through the same sequence? Doesn't this cause PCR artefacts?
The last 14 nucleotides of P5 and P3 solexa primers are identical. Interestingly, that doesn’t create any artefacts in our hands.