GSM3559354: B2B10GROseq-TE2; Homo sapiens; OTHER
Plates were washed in PBS and cells were scraped in Lysis Buffer prior to isolation of nuclei. Nuclear run-on reactions were performed in the presence of Br-UTP, then total RNA was isolated using TRIzol Reagent. RNA was hydrolyzed with NaOH to yield fragments ~200 bp long and then treated with DNase prior to two sequential nascent RNA enrichment steps performed by anti-Br-UTP immunoprecipitation. A random-priming library preparation kit (NEBNext Ultra Directional RNA Library Prep Kit for Illumina (New England Biolabs)) was used according to the Protocol for use with NEBNext rRNA Depletion Kit provided by the manufacturer to generate uniquely barcoded libraries for sequencing. Libraries were size-selected for a target size of ~300 bp and sent to an Illumina HiSeq instrument for 50 bp single-end sequencing.
GEO Accession
GSM3559354