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First time posting and first effort at sequencing so please bear with me.
I am trying to use Illumina's small RNA kit to perform PAR-CLIP. I saw radio-labelled products at the correct MW for my proteins that was dependent on being UV treated so I think the cross linking worked. I was able to recover RNA from these too (saw an RNA pellet that was hot!). I didn't quantify the RNA at this point because the samples were pretty radioactive but reading some posts I'm wondering if I should have.
I then moved on to the kit. I did the 11 cycles suggested by the kit but couldn't see anything on the bioanalyzer above 120bp. I decided at this point I hadn't done enough cycles so repeated the RT and PCR (using some more of the ligated sample - you don't use it all going into the RT) this time with 18 cycles.
I could now see something on the bioanalyser in all 3 of my samples - predominantly 125-128 bp. There was also some larger products - maybe up to 200 but these were much smaller.
On a gel again it looked like a lot of 120bp but also a significant amount of a larger product - 220bp approx. I don't this was reflexed on the bioanalyzer.
I gel purified from 140 up to 200 (just below the other larger product) but also cut this larger product out too and ran both these at the end.
I've attached the 11 cycle, 18 cycle and gel for one of my samples then the final trace for all 3 - the 140-200 slice I cut. The larger product indicated 187-9 bp .
My guess was that maybe I have adapter-adapter dimers amplifying which was giving a 12x bp product.
First question - how do my final traces look. Struggling for guidance !
Second question is how to reduce the presumably adaptor dimer product I see? - use less 3' adaptor?, On bead 3' ligation?, Purification of the ligated product after ligating both adaptors? I've read about heat inactivating the ligase in a similar manner to the NEBNext Kit? - is this after the first of second one and how would this help.
Any help or guidance would be greatly appreciated. Kinda gone in blind and have been lurking on here for general tips but though a post might be more helpful - sorry for the length.
I am trying to use Illumina's small RNA kit to perform PAR-CLIP. I saw radio-labelled products at the correct MW for my proteins that was dependent on being UV treated so I think the cross linking worked. I was able to recover RNA from these too (saw an RNA pellet that was hot!). I didn't quantify the RNA at this point because the samples were pretty radioactive but reading some posts I'm wondering if I should have.
I then moved on to the kit. I did the 11 cycles suggested by the kit but couldn't see anything on the bioanalyzer above 120bp. I decided at this point I hadn't done enough cycles so repeated the RT and PCR (using some more of the ligated sample - you don't use it all going into the RT) this time with 18 cycles.
I could now see something on the bioanalyser in all 3 of my samples - predominantly 125-128 bp. There was also some larger products - maybe up to 200 but these were much smaller.
On a gel again it looked like a lot of 120bp but also a significant amount of a larger product - 220bp approx. I don't this was reflexed on the bioanalyzer.
I gel purified from 140 up to 200 (just below the other larger product) but also cut this larger product out too and ran both these at the end.
I've attached the 11 cycle, 18 cycle and gel for one of my samples then the final trace for all 3 - the 140-200 slice I cut. The larger product indicated 187-9 bp .
My guess was that maybe I have adapter-adapter dimers amplifying which was giving a 12x bp product.
First question - how do my final traces look. Struggling for guidance !
Second question is how to reduce the presumably adaptor dimer product I see? - use less 3' adaptor?, On bead 3' ligation?, Purification of the ligated product after ligating both adaptors? I've read about heat inactivating the ligase in a similar manner to the NEBNext Kit? - is this after the first of second one and how would this help.
Any help or guidance would be greatly appreciated. Kinda gone in blind and have been lurking on here for general tips but though a post might be more helpful - sorry for the length.