GSM4566727: MmGtsf1-Strep in Sf9 - rip1_1; Spodoptera frugiperda; RIP-Seq
Cells were lysed by sonication in 50 mM Tris, pH 8.0, 100 mM KCl, 1 mM DTT, then purified by affinity chromatography using Strep-Tactin resin (IBA). The purified Gtsf1 protein and co-purifying nucleic acids were eluted in lysis buffer supplemented with 5 mM d-desthiobiotin. The eluted material was further purified by ion exchange chromatography (MonoQ column) and nucleic acid containing fractions were taken for library construction. Small RNA libraries were prepared using the SMARTer smRNA-Seq Kit for Illumina sequencing (Takara). In addition to the co-purifying RNAs, total Sf9 RNAs were similarly processed as input controls. Size-selection was performed using Blue Pippin 2% agarose gel cassettes (Sage Science). All libraries were assessed by fluorometric quantification (Qubit 3.0) and by Bioanalyzer chip-based capillary electrophoresis. Libraries were pooled in equimolar ratios according to their quantification by Bioanalyzer. Single-end reads with two 8-basepair barcodes were generated on an Illumina NextSeq resulting in approximately 10 million reads per library.
GEO Accession
GSM4566727