Hi everyone,
I am new to RNAseq analysis and am making preparations for the design of an RNAseq experiment to determine the differentially expressed long non-coding RNAs between virus-infected and uninfected cells in culture.
I would greatly welcome comments on what is optimal for the following or indeed what else I should be considering:
1. RNA prep with DNase I (on-column or in solution digestion?)
I am using Direct-zol RNA prep from Zymo Reserach with on-column DNAase I digestion.
2. the library prep
Illumina TruSeq stranded mRNA library prep kit is the plan. Is oligo-dT capture okay?
3. what platform is best?
HiSeq and fast run mode was suggested
4. what number of reads/per biological replicate is needed
Is 3 biological replicates and 10 M reads per biological replicate enough?
5. single read or paired end reads
Single end is okay?
6. read length
>75?
I would be extremely grateful if members could please recommend what kits are best suited to the task as well: Illumina kits? (library preparation kit and ribosomal RNA depletion?) to maximise the chances of success to detect differentially expressed long non-coding RNA transcripts.
Many thanks for your time and expertise and it really is greatly appreciated,
Fitzcarraldo
I am new to RNAseq analysis and am making preparations for the design of an RNAseq experiment to determine the differentially expressed long non-coding RNAs between virus-infected and uninfected cells in culture.
I would greatly welcome comments on what is optimal for the following or indeed what else I should be considering:
1. RNA prep with DNase I (on-column or in solution digestion?)
I am using Direct-zol RNA prep from Zymo Reserach with on-column DNAase I digestion.
2. the library prep
Illumina TruSeq stranded mRNA library prep kit is the plan. Is oligo-dT capture okay?
3. what platform is best?
HiSeq and fast run mode was suggested
4. what number of reads/per biological replicate is needed
Is 3 biological replicates and 10 M reads per biological replicate enough?
5. single read or paired end reads
Single end is okay?
6. read length
>75?
I would be extremely grateful if members could please recommend what kits are best suited to the task as well: Illumina kits? (library preparation kit and ribosomal RNA depletion?) to maximise the chances of success to detect differentially expressed long non-coding RNA transcripts.
Many thanks for your time and expertise and it really is greatly appreciated,
Fitzcarraldo
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