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Dear all,
I am a research fellow at Birmingham UK, looking to study meta-transcriptomics from colon biopsies in patients with colitis. I am very new to this but have had some sequencing experience with 16s and metagenomics (including library preparation). I am still trying to get my head around the different steps in order to successfully prepare cDNA libraries and I would be grateful for any help anyone can provide. So far I am able to successfully get RNA out of colon biopsies with a RIN > 8 and around 300ng/ul (total volume 50ul) with the current protocol that I am using. I have a few questions please:
1) The next step from what I understand is to remove host RNA and then enrich bacterial mRNA by removing rRNA. I have got two kits for these - Ambion MicrobeEnrich and MicrobeExpress for these steps respectfully, but havent yet used them.
2) As I don't expect there to be a lot of bacterial mRNA should I use the Smarter standed RNA-Seq kit to make cDNA libraries?
3) How many samples can I multiplex in a run? - We have a Hiseq and Miseq sequencing platforms. Is it just a matter of sequencing a few and seeing how much depth I have got?
Thank you again for any help you can provide!
I am a research fellow at Birmingham UK, looking to study meta-transcriptomics from colon biopsies in patients with colitis. I am very new to this but have had some sequencing experience with 16s and metagenomics (including library preparation). I am still trying to get my head around the different steps in order to successfully prepare cDNA libraries and I would be grateful for any help anyone can provide. So far I am able to successfully get RNA out of colon biopsies with a RIN > 8 and around 300ng/ul (total volume 50ul) with the current protocol that I am using. I have a few questions please:
1) The next step from what I understand is to remove host RNA and then enrich bacterial mRNA by removing rRNA. I have got two kits for these - Ambion MicrobeEnrich and MicrobeExpress for these steps respectfully, but havent yet used them.
2) As I don't expect there to be a lot of bacterial mRNA should I use the Smarter standed RNA-Seq kit to make cDNA libraries?
3) How many samples can I multiplex in a run? - We have a Hiseq and Miseq sequencing platforms. Is it just a matter of sequencing a few and seeing how much depth I have got?
Thank you again for any help you can provide!