Back to basics: how do I perform RNA-seq? Here's one way

Fresh out of my lab notebook, this is how I conducted RNA-seq with success from human/mouse FACS-purified cells. If you're a new user, you may ask NEB for free trial kits for all reagents listed in this protocol!

  1. Purify RNA using Monarch® Total RNA Miniprep Kit (T2010) https://international.neb.com/products/t2010-monarch-total-rna-miniprep-kit#Product%20Information
  2. Perform rRNA depletion using NEBNext® rRNA Depletion Kit v2 (Human/Mouse/Rat) with RNA Sample Purification Beads (E7405) https://www.neb.uk.com/products/neb-catalogue/ngs-sample-prep/nebnext-lt;sup-gt;-amp;reg;-lt;-sup-gt;-rrna-depletion-kit-v2-(human-mouse-rat)-with-rna-sample-purification-beads - I recommend this approach over mRNA enrichment, as it allows you to recover noncoding RNAs from your bulk RNA-seq data, which will provide more information on gene regulation
  3. Prepare the RNA-seq library using NEBNext® Ultra™ II Directional RNA Library Prep Kit for Illumina® (E7760) https://international.neb.com/products/e7760-nebnext-ultra-ii-directional-rna-library-prep-kit-for-illumina#Product%20Information

I sequenced my RNA-seq libraries generated from this protocol at 25 million reads per sample on HiSeq 4000 using PE75. You can do what suits you, but this is one suggestion. Enjoy! Happy to address any questions here or on Sci Find Discord


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Stefani Robnett3 months ago

This looks super useful Di! Thanks for the references to these other protocols... Re-sharing this with the other forums :)

Di Hu3 months ago

Thank you Stefani! I'm asked how to perform this technique all the time. This protocol allows you to try everything for free as a first time user of these specific NEB kits. Since the entire process is sourced from NEB, they are also happy to help troubleshoot any issues along the way. I'm happy to help as well!

Nick Gervais3 months ago

Super helpful! We usually send samples out for RNA-Seq and it's super expensive. What does the RNA-Seq library look like after the final step? We don't have a HiSeq 4000 on our floor but I'm wondering if the library would be stable enough to ship out for sequencing...

Di Hu3 months ago

Thank you Nick! Here's how the final library should look like: https://drive.google.com/file/d/1N8YXf3NtEb-b8SEsXTN0kbPzM1AisGim/view?usp=sharing If it doesn't look like this, feel free to send me some example traces of what you have whenever (di@athenadao.co) and I can suggest ways to troubleshoot. I don't have a HiSeq either. I send it out to sequencing at Novogene in my case, as I'm in London, UK. I usually send it at room temperature, or with an ice pack for overnight shipping. The stability should be fine, as the final library is DNA. I've not had issues doing this for the past 4 years

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