GSM7719587: EU-seq WT ERα OE  Repeat 1; Homo sapiens; RNA-Seq
For ChIP-seq experiment, genomic DNA was isolated as per standard ChIP-seq protocol. For fRIP-seq, RNA crosslinked to ERα were pulldown using anti ERα followed by purification of the total RNA using trizol. For EU-seq cells were chased with EU for 45 mins and total RNA was isolated using trizol. Further EU labelled RNA was biotinylated using Click Chemistry reaction and pulldown using Streptavidin beads using standard protocol. The pulldown RNA was extracted using Trizol and used for library preparation. ChIP-seq library preparation using the NEBNext® Ultra™II DNA Library Prep kit with Sample Purification Beads (Catalogue-E7103L) following the manufacturer's protocol. The fRIP-seq purified RNA was used for preparing RNA-seq libraries, using the NEBNext rRNA Depletion Kit v2 and NEBNext Ultra II Directional RNA Library Prep, following the manufacturer's protocol. For EU-seq the nasecent RNA was converted to cDNA using the SuperScript™ IV First-Strand Synthesis System (catalogue number: 18091050). The second strand of cDNA was synthesized using the NEBNext® Ultra™ II Directional RNA Library Prep with Sample Purification Beads (Catalogue no-E7765L). The double-stranded DNA was further fragmented, and the library was prepared using the NEBNext® Ultra™ II FS DNA Library Prep Kit for Illumina (E7805L), following the manufacturer's protocol.