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Old 03-16-2017, 01:10 PM   #2
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Location: New Haven

Join Date: Mar 2016
Posts: 8

Most of these questions are going to be answered based on your budget and the scale of your experiment.

If you want to look for rare transcripts, higher sequencing depth per sample will be more important. If you plan on doing lots of samples with many replicates then you may be less interested in reads per sample.

If your goal is purely to look at lncRNA, oligo-dT capture may not be ideal as many - perhaps even most - lncRNA are not polyadenylated. I've never tried it but ribosomal depletion is a popular alternative.

If you dont care about costs or time and you're using a highseq 1500 or 2500, then high output mode > rapid run mode because you'll get more reads per lane. There are newer "better" Illumina platforms but I dunno what's available for you or if your institution has troubleshooted the new platforms well enough to trust them.

Paired-end sequencing might be more ideal for you because lncRNA often have hella splice variants and you'll want to read through more than one exon to see those.

If possible, first do a trial experiment and analyze the sequence data before preparing all of the libraries to avoid wasting lots of money building bad libraries or sequencing lots of ugly data. You can even sequence this trial run at a higher depth than usual, then see what happens when you down-sample to different read depths to try to optimize sequencing saturation.
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