GSM3613783: EVneg; Homo sapiens; RNA-Seq
Cells were detached from the culture dish with citric saline (270mM KCl, 30mM sodium citrate), passed through a 40um filter and counted using a hemocytometer to collect 500.000 cells. Total RNA was extracted using the Nucleospin RNA kit (Macherey Nagel, 740955.5) according to manufacturer's instructions. DNase treatment was used to exclude genomic DNA. Libraries were constructed by the Centre for Applied Genomics in Toronto (ON, Canada). RNA-seq libraries were prepared using the NEBNext Poly(A) mRNA Magnetic Isolation Module (New England Biolabs, E7490) to enrich for poly(A) mRNA and the NEBNext Ultra Directional RNA Library Prep Kit for Illumina (New England Biolabs, E7420), according to manufacturers' specifications. Libraries were indexed using the NEBNext Multiplex Oligos 1-8 for Illumina (Index Primers Set 1; New England Biolabs, E7335), according to manufacturers' specifications.