Hi all,
I'm trying to perform a scRNA-like library prep in bacteria, so I cannot take advantage of the polyT reverse transcription primers. Instead, I'm using tagged random hexamers/decamers. I also need to use a relatively long template switching oligo that is about 90 nt and contains 20 random bases as an UMI.
So far, I haven't had a lot of luck with high efficiencies (I would estimate I'm generating less than one cDNA fragment per cell).
I'm working on optimizing everything, but I'm hopeful that someone has some insight into what is the limiting step? Lysis is definitely not an issue, but RNA is possibly modified by formaldehyde. RNase contamination is very unlikely
My RT reaction
Superscript II (10U/uL, standard SSII protocol -> is it detrimental to use too much RTase relative to RNA input?)
1 mM dNTP
1 uM TSO
2.5 uM Random hexamers
10 mM DTT
20 ug/mL BSA
Enzymatics RNase inhibitor
Leave 1 hour at room temperature (to facilitate hybridization of random hexamers), then overnight at 37 degC. I know this is unconventional, but my system is quite a bit different than normal...
I appreciate any and all advice/help!
I'm trying to perform a scRNA-like library prep in bacteria, so I cannot take advantage of the polyT reverse transcription primers. Instead, I'm using tagged random hexamers/decamers. I also need to use a relatively long template switching oligo that is about 90 nt and contains 20 random bases as an UMI.
So far, I haven't had a lot of luck with high efficiencies (I would estimate I'm generating less than one cDNA fragment per cell).
I'm working on optimizing everything, but I'm hopeful that someone has some insight into what is the limiting step? Lysis is definitely not an issue, but RNA is possibly modified by formaldehyde. RNase contamination is very unlikely
My RT reaction
Superscript II (10U/uL, standard SSII protocol -> is it detrimental to use too much RTase relative to RNA input?)
1 mM dNTP
1 uM TSO
2.5 uM Random hexamers
10 mM DTT
20 ug/mL BSA
Enzymatics RNase inhibitor
Leave 1 hour at room temperature (to facilitate hybridization of random hexamers), then overnight at 37 degC. I know this is unconventional, but my system is quite a bit different than normal...
I appreciate any and all advice/help!
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