Trizol extraction of total RNA was performed according to the manufacturer's instructions. External spike-in RNAs (ERCC ExFold RNA Spike-In kit, Ambion, 4456739) were added based on cell number. See manuscript for details. For NET-seq, the library preparation is performed as described by Churchman and Weissman (2011, 2012) with modifications. For 30 RNA ligation, a pre-adenylated DNA linker with a mixed random hexameric barcode sequence at its 50 end is used. cDNA containing the 30 end sequences of a subset of mature and heavily sequenced snRNAs, snoRNAs, rRNAs, and mitochondrial tRNAs are specifically depleted using biotinylated DNA oligos, as described by Ingolia et al.
GEO Accession
GSM2090429