GSM3834685: Bcell_PROCap_WT2_39537; Mus musculus; OTHER
Following cell membrane lysis with hypotonic buffer, nuclei were isolated via sucrose gradient density centrifugation. PRO-cap was performed as described (Kwak et al., 2013 and Mahat et al., 2016). Briefly, 20 million nuclei were used per reaction, and the run-on was performed for 5 minutes with biotin-NTPs  followed by extraction of RNA with Trizol reagent (Thermo Fisher, 15596026) and subsequent enrichment of biotin-labeled RNA with anti-BrU beads. After 3' end repair, Illumina-compatible 3' end RNA adapters were ligated. All free 5' end were dephosphorylated and capped 5'ends were enzymatically treated to remove the cap to ligate the Illumina-compatile 5' end RNA adapters. After Streptavidin enrichment of biotin labeld RNA, the library was reverse transcribed and amplified.
GEO Accession
GSM3834685