Hi, I would appreciate a little help on the following situation.
I have bacterial total RNA that I separate into several samples and want to sequence afterward. From my controls I know that each of those samples contains a very different part of the total RNA (e.g. some samples don't contain rRNA).
In order to still be able to normalize my sequencing results, I most likely cannot use the usual normalization methods that assume that most of the transcripts don't differ between samples.
Therefore, I think a spike-in is an option allowing me to normalize my data without the "help" of my actual sample. For this, I want to use the ERCC spike-in and use RUV for normalization afterward.
This is where I'm at at the moment. My current plan is to add the spike-in after RNA extraction (prior is not possible) and then continue with rRNA depletion, fragmentation and library prep (most likely using polyA-ligation). However, I'm not sure, how exactly the best way of adding the spike-in in this case would be. The "official" way is to measure the RNA concentration (which is not the precisest thing anyway) and then add a set amount of spike-in to each sample. As far as I understand, the reason to do so is to be able to calculate the dynamic range of your sequencing afterward. However, I'm not sure I'm interested in that. Is it possible to add the same amount of spike-in to each sample and then normalize according to the read numbers of the spike-in in each sample? What would be the (dis)advantage in doing so?
I have bacterial total RNA that I separate into several samples and want to sequence afterward. From my controls I know that each of those samples contains a very different part of the total RNA (e.g. some samples don't contain rRNA).
In order to still be able to normalize my sequencing results, I most likely cannot use the usual normalization methods that assume that most of the transcripts don't differ between samples.
Therefore, I think a spike-in is an option allowing me to normalize my data without the "help" of my actual sample. For this, I want to use the ERCC spike-in and use RUV for normalization afterward.
This is where I'm at at the moment. My current plan is to add the spike-in after RNA extraction (prior is not possible) and then continue with rRNA depletion, fragmentation and library prep (most likely using polyA-ligation). However, I'm not sure, how exactly the best way of adding the spike-in in this case would be. The "official" way is to measure the RNA concentration (which is not the precisest thing anyway) and then add a set amount of spike-in to each sample. As far as I understand, the reason to do so is to be able to calculate the dynamic range of your sequencing afterward. However, I'm not sure I'm interested in that. Is it possible to add the same amount of spike-in to each sample and then normalize according to the read numbers of the spike-in in each sample? What would be the (dis)advantage in doing so?
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