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Hi everyone, this is my first foray into data analysis for RNAseq and I want to get a general idea of whether or not I'm doing this correctly.
I conducted RNA pulldowns and based on my in vitro biochemical data as well as Bioanalyzer results I believe tRNAs may actually be some of the RNAs I'm expecting.
What I want to do now is to see if there are any tRNA reads in my data sets and how to get the number reads for different tRNA species. I know from reading posts here that this may be tricky due to base modifications and biased reads due to secondary structure.
Questions for mapping: I've mapped my reads with Tophat allowing reads to map to 50 genomic loci (based on UCSC's genomic tRNA database the highest number of loci for a given tRNA isoacceptor (Ala) is 43). Is this the correct way to go about getting good mapping for tRNAs? Does anyone have thoughts on another way to go about this
After mapping I'm going to run HTSeq-count using a custom tRNA GTF. As far as I can tell that's what I should do...or am I missing something?
I'd greatly appreciate any help you guys can offer!
I conducted RNA pulldowns and based on my in vitro biochemical data as well as Bioanalyzer results I believe tRNAs may actually be some of the RNAs I'm expecting.
What I want to do now is to see if there are any tRNA reads in my data sets and how to get the number reads for different tRNA species. I know from reading posts here that this may be tricky due to base modifications and biased reads due to secondary structure.
Questions for mapping: I've mapped my reads with Tophat allowing reads to map to 50 genomic loci (based on UCSC's genomic tRNA database the highest number of loci for a given tRNA isoacceptor (Ala) is 43). Is this the correct way to go about getting good mapping for tRNAs? Does anyone have thoughts on another way to go about this
After mapping I'm going to run HTSeq-count using a custom tRNA GTF. As far as I can tell that's what I should do...or am I missing something?
I'd greatly appreciate any help you guys can offer!