Dear all,
Just wanted to share with you some possible modifications i will be implementing in the ScriptSeq RNA v2 protocol for sequencing using the V3 chemistry.
1- I was thinking to reduce the RNA fragmentation time to 3 or 4 min at 85 deg (down from 5min at 85 deg).
2- Increase cDNA synthesis time using random hexamers to 60min at 42 deg using AMV (up from 20min at 42 deg)
3- cDNA purification using 0.9x beads (instead off 1.8x previously) - too rough at this step?
4- RNA seq lib purification using 0.85x beads (instead off 1x previously)
What do you think about the following modifications?
I have been having problems lately using the protocol as it is with the v3 chemistry (2x300bp read length).
My library had an average fragment length of 400bp ending resulting in a mean read length of 180bp (which is quite short!).
I just wanted to make sure those steps make sense.
Best,
E
Just wanted to share with you some possible modifications i will be implementing in the ScriptSeq RNA v2 protocol for sequencing using the V3 chemistry.
1- I was thinking to reduce the RNA fragmentation time to 3 or 4 min at 85 deg (down from 5min at 85 deg).
2- Increase cDNA synthesis time using random hexamers to 60min at 42 deg using AMV (up from 20min at 42 deg)
3- cDNA purification using 0.9x beads (instead off 1.8x previously) - too rough at this step?
4- RNA seq lib purification using 0.85x beads (instead off 1x previously)
What do you think about the following modifications?
I have been having problems lately using the protocol as it is with the v3 chemistry (2x300bp read length).
My library had an average fragment length of 400bp ending resulting in a mean read length of 180bp (which is quite short!).
I just wanted to make sure those steps make sense.
Best,
E
Comment