Hi Maria, in my experience with RNASeq on Tomato plants, the RIN values as calculated by the Tapestation software are unreliable for plants, due to the smaller peaks for the chloroplast RNA being interpreted as degradation. Having said that, looking at your electropherograms there does seem to be some real degradation, especially in the bottom-left sample. I have produced very good RNASeq libraries using samples that looked similar to the other three, though. What fragmentation method are you using? 

Hi Tom,

Thanks a lot for your quick reply and comments. For the library preparation I am using the TruSeq Stranded Total RNA kit with Ribo-Zero plant kit from Illumina in which after depleting the RNA ribosomal in my samples, I am incubating the RNA with the Elute, Prime, Fragment Mix at 94C for 8 minutes, less if my samples are degraded. My concern is whether I should decrease the fragmentation time to 7 or less according to the degradation levels I am having...

Thanks

Maria

I have never decreased the fragmentation time in the "Elution 2-FRAG-PRIME" thermal cycler program step, and always produced libraries of good quality. But I have never tried library preparation using samples with a level of degradation equal to that of the worst quality sample you provided. Have you been in touch with Illumina about this? I always find that their customer service/troubleshooting is very helpful. You could just give them a call and find out what they think.