Hi there,
We are sequencing RNA samples with the following pipeline:
1. globin clear treatment
2. making cDNA on a Caliper robot
3. prepare cDNA library prep for sequencing on Caliper robot
After step 3 we always check the products on a labchipGX. For most samples we see a peak around 300 bp. But for some samples (depending on the plate) we also see an additon peak around 1000-1500 bp. To my knowledge there could be three reasons (fragmentation incomplete, AMpure beads carryover, bubble products). I tried to denature and very slowly renature the products, but still see the binomial pattern. Also AMpure carryover is exlcuded as I use a magnet for dilution. Fragmentation can also be exluded as all samples undergo the same fragmentation procedure.
Did anyone else see binominal peak patterns after the library prep. And how did you solve it?
Many thanks!
We are sequencing RNA samples with the following pipeline:
1. globin clear treatment
2. making cDNA on a Caliper robot
3. prepare cDNA library prep for sequencing on Caliper robot
After step 3 we always check the products on a labchipGX. For most samples we see a peak around 300 bp. But for some samples (depending on the plate) we also see an additon peak around 1000-1500 bp. To my knowledge there could be three reasons (fragmentation incomplete, AMpure beads carryover, bubble products). I tried to denature and very slowly renature the products, but still see the binomial pattern. Also AMpure carryover is exlcuded as I use a magnet for dilution. Fragmentation can also be exluded as all samples undergo the same fragmentation procedure.
Did anyone else see binominal peak patterns after the library prep. And how did you solve it?
Many thanks!