Illumina Sequencing

Are the solexa primers that are given in the paper designed for a paired-end sequencing?

Yes, but we use them for single-end sequencing.

My library is ready, so I’d like to know whether need customized sequencing primer or just the standard primer for Illumina sequencing.

The standard sequencing primer is used.

What kind of sequencing was employed? Does a standard Illumina sequencing flow need to be customized?

We use 75 or 100 cycles single-end sequencing with HiSeq. Standard protocol.

How many reads do you normally aim for per sample?

10 million if the quality measures were good (low PCR cycle number, expected size distribution of amplicons). After initial round of sequencing, we often then resequence the library if we see the ratio of cDNA/read count is close to 1 (i.e., most reads have unique UMIs).

Do you use CLIP-only samples in a flow cell or do you mix with other samples? Our facility doesn't like mixing "unusual" samples together.

We had designed our custom indexes in such a way that they are different from indexes of all other methods and kits used by the facility at the Crick institute. So we let our facility to decide if they wish to mix with other samples, and in most cases they do. They haven't seen any problems when mixing our 'unusual' iCLIP libraries with others, it doesn't affect the quality of other samples.

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