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!
- Purify RNA using Monarch® Total RNA Miniprep Kit (T2010) https://international.neb.com/products/t2010-monarch-total-rna-miniprep-kit#Product%20Information
- 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
- 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