title: GSM2396015: 5'GRO-seq without fragmentation; Homo sapiens; OTHER
library_construction_protocol: Two 5’-GRO-seq experiments were carried out by variation of the procedure described in Duttke et al. (2015) and Hetzel et al. (2016). We were curious with regard to whether the RNA fragmentation step would affect the 5'-GRO-seq data, and we therefore performed one experiment with the RNA fragmentation and other experiment without the RNA fragmentation. As shown in Supplemental Fig. S2, we obtained similar results from the two experiments, and thus used both datasets for our analyses. For each experiment, nuclear run-on reactions were performed with 107 MCF-7 nuclei in the presence of BrUTP (Sigma Aldrich) for labeling of the RNA. Trizol LS (Fisher Scientific) was used to terminate the reaction and to extract the RNA. For 5’-GRO-seq with fragmentation, RNA was hydrolyzed by Zn(II)-mediated fragmentation (Ambion). BrU-labeled transcripts were immunoprecipitated with anti-BrdU agarose beads (Santa Cruz Biotech) and extracted with Trizol LS. The resulting RNA was incubated with T4 polynucleotide kinase (NEB) in low pH buffer followed by treatment with calf intestinal alkaline phosphatase (CIP; NEB) to achieve 5’ and 3’ dephosphorylation. A second BrU enrichment was performed, and 5’-monophosphate-RNAs were removed with Terminator 5’-phosphate-dependent exonuclease (Epicentre), as described in Hetzel et al. (2016). The RNA was subjected to a second round of dephosphorylation with CIP (NEB). Decapping and library preparation were performed as described in Hetzel et al. (2016). For 5’-GRO-seq without RNA fragmentation, the run-on step was followed by RNA extraction with Trizol. BrU-labeled transcripts were immunoprecipitated with anti-BrdU agarose beads. RNA was dephosphorylated with CIP (NEB), and subjected to a second round of BrU enrichment that was followed by a second round of CIP (NEB). Decapping and library preparation were performed as described in Hetzel et al. (2016). It might be noted that these experiments involve the ligation of an adapter (GTTCAGAGTTCTACrArGUrCrCrGrArCrGrAUrC) to the 5' end of the transcripts. The ligation step has been found to exhibit sequence bias (Jayaprakash et al. 2011). Hence, it might alter the relative amounts of different transcripts, but would not affect the accuracy of the mapping of the 5' ends of the transcripts. The Small RNA Library Prep Set (New England Biolabs) was used for final library preparation with modifications as described in Hetzel et al. 2016
attributes.tag: GEO Accession
attributes.value: GSM2396015