Detection of L1Hs Retrotransposon RNA

HCT116 cells were transfected with 5 nM siRNA (as indicated in Fig. 2h) then, 48 h later, were either transfected with a plasmid encoding L1Hs and driven by a minimal EF1alpha (without an intron) promoter or mock transfected. Twenty-four hours later (72 h post siRNA transfection), cells were trypsinized and resuspended with a buffer containing 0.5% Igepal CA-630, essentially as described in ref. ^{57 (link)}. The cytoplasmic fraction was purified with RNA Clean & Concentrator columns (Zymo), 2 µg of which was loaded onto 1.2% agarose gel and electroblotted to a nylon membrane. DIG-labelled probes against ORF2 were prepared with in vitro transcription (see Supplementary Table 2 for primers) and probe hybridization, washes and imumunodetection were carried out as described in the manual of the DIG Northern Starter Kit (Roche, no. 12 039 672 910).