GSM1523196: E2-siCTL_repeat2_GROseq (v.s. E2-siNCAPD3_GROseq); Homo sapiens; RNA-Seq
MCF7 cells were subjected to nuclear run-on for 5minutes at 30 degree with BrU labeling. The run-on RNAs were pull down by BrU beads and subjected to library preparation for deep sequencing. We followed a previous published protocol (Ingolia et al., 2009 Science; Wang et al., 2011 Nature; Li et al., 2013 Nature). Briefly, the run-on RNA were first added with a polyA tracts by RNA polyA polymerase. This polyA tail enables the run-on RNA to be reverse transcribed into single strand cDNA by Reverse Transcriptase. The cDNA was circularized by CircLigase (Epicentre) and re-linearized by Ape1, subsequently subjected to PCR amplification and deep sequencing.