Hello everybody
I'm going to prepare RNAseq libraries from cassava. First I did was purify mRNA from my samples using polydT(25) beads (life technologies). Then using 5x First Strand Buffer (Invitrogen) I incubated the samples at 94C for different times (2, 5 and 7 minutes). I sent this samples to Bioanalyzer before first strand cDNA synthesis and the profile is really weird. There's a peak around 200 bp (so the fragmentation seems to be good, especially 5 and 7 min: 2 was not enough) but the peak itself and the baseline are very noisy (distorted). I've read that can be a problem with the chip itself, but also because of not enough washing after the fragmentation. The thing is that the protocol I'm following doesn't not incluse a washing after this fragmentation. What do you think about this profile?. Any suggestion of what to do with these samples?. Thanks!
I'm going to prepare RNAseq libraries from cassava. First I did was purify mRNA from my samples using polydT(25) beads (life technologies). Then using 5x First Strand Buffer (Invitrogen) I incubated the samples at 94C for different times (2, 5 and 7 minutes). I sent this samples to Bioanalyzer before first strand cDNA synthesis and the profile is really weird. There's a peak around 200 bp (so the fragmentation seems to be good, especially 5 and 7 min: 2 was not enough) but the peak itself and the baseline are very noisy (distorted). I've read that can be a problem with the chip itself, but also because of not enough washing after the fragmentation. The thing is that the protocol I'm following doesn't not incluse a washing after this fragmentation. What do you think about this profile?. Any suggestion of what to do with these samples?. Thanks!
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