GSM1654863: LacZ RNAi sm RNA-seq rep3; Drosophila melanogaster; ncRNA-Seq
Total RNA from 8 x 106 Drosophila Dmel-2 tissue culture cells was isolated using QIAzol Lysis Reagent (Qiagen). Total RNA was fractionated into large (>200 nts) and small (<200 nts) fractions using RNeasy Mini spin columns and RNeasy MinElute spin columns, respectively (Qiagen). DNA was removed from the large fraction by on-column DNase digestion (Qiagen). 28S, 18S and 5S rRNAs were depleted from 5 µg of each large RNA fraction using the Ribo-Zero Magnetic Kit (Epicentre). 2S rRNA was depleted from the small RNA fraction according to Seitz et al.  (SEITZ et al. 2008) with the following modifications. 0.1 nMoles 2S rRNA complementary oligo were bound to 500 µg streptavidin beads in 1 mL 0.5x SSC for one hour at 4oC. The beads were then washed 5X in 0.5X SSC followed by 5 minute incubation at 65oC to remove secondary structure. 2 µg of the small RNA fraction were diluted to 12.5 ng/µl and 160 µl were added to the bead slurry. RNA-seq libraries were prepared in triplicate from 35 ng of the rRNA-depleted large RNA fraction using the NEBNext Ultra Directional RNA Library Prep Kit for Illumina (NEB). smRNA-seq libraries were prepared in triplicate from ~475 ng of the 2S rRNA-depleted small RNA fraction using the NEBNext Small RNA Library Prep Set for Illumina (NEB).
GEO Accession
GSM1654863