title: GSM4125143: Long_RNA_fragments; synthetic construct; OTHER
library_construction_protocol: Illumina adapter sequences were incorporated to the libraries using PCR. The DNA sequences encoding pri-miRNAs were amplified from human HCT116 genomic DNA using specific primers. Pri-miRNAs were synthesized by in vitro transcription (IVT),  visualized on 10% UREA-PAGE and gel-eluted followed by isopropanol precipitation. The NLS Microprocessor complex (composed of NLS-D3 fragment of DROSHA and DGCR8) or minimal Microprocessor complex (composed of D3 fragment of DROSHA and G4 dimer of DGCR8)  was co-expressed and purified in HEK293E cells. Purified Microprocessor complex was used in in vitro processing assay for pri-miRNAs. After visualized in 10% UREA-PAGE, the fragments from the reaction mixture which are longer than pre-miRNAs, 5p and 3p basal segments were sliced from the gel and cloned to construct a DNA library for next generation sequencing.
identifiers.namespace: GEO
identifiers.id: GSM4125143