title: RNA-seq Microglia: E4_17a_2
design: For RNA isolation, frozen cell pellets were resuspended in 1ml of Trizol (Invitrogen, Carlsbad, CA) and total RNA was purified following the manufacturers instructions. RNA quality was assessed using the Agilent Bioanalyzer platform at the USC Genome Core using the RNA integrity number. Then, 500ng of total RNA was subjected to ribosomal-RNA depletion using the NEBNext rRNA Depletion kit (E7850L, New England Biolabs, Ipswich, MA), according to the manufacturers protocol. Strand specific RNA-seq libraries were then constructed using the SMARTer Stranded RNA-seq kit (634485, Clontech, Kusatsu, Shiga, Japan), according to the manufacturers protocol. Libraries were quality controlled on the Agilent Bioanalyzer 2100 platform at the USC Genome Core before multiplexing the libraries for sequencing. Paired-end 150-bp reads were generated on the Illumina NovaSeq platform at the Novogene Corporation (Sacramento, CA).