title: GSM5288460: KD_MB6_A; Mus musculus; OTHER
library_construction_protocol: ChRO-seq library preparation: ChRO-seq libraries were prepared following a recent protocol (Mahat et al., 2016). We prepared some libraries to achieve single nucleotide resolution for the Pol II active site. In these cases, the chromatin pellet was incubated with 2x run-on buffer (10 mM Tris-HCl, pH 8.0, 5 mM MgCl2,1 mM DTT, 300 uM KCl, 20 uM  Biotin-11-ATP (Perkin Elmer, NEL544001EA), 200 uM Biotin-11-CTP (Perkin Elmer, NEL542001EA), 20 uM Biotin-11-GTP (Perkin Elmer, NEL545001EA), 200 uM Biotin-11-UTP (Perkin Elmer, NEL543001EA)) for 5 minutes at 37 C. In some libraries we modified the run-on buffer to extend the length of reads for more accurate allelic mapping at the expense of single nucleotide resolution for the Pol II active site. In these cases, the run-on reaction was performed using a different ribonucleotide composition in the nuclear run-on buffer (10 mM Tris-HCl, pH 8.0, 5 mM MgCl2,1 mM DTT, 300 mM KCl, 200 μ M  ATP (New England Biolabs (NEB), N0450S), 200 μ M  UTP, 0.4 μ M CTP, 20 μ  M Biotin-11-CTP (Perkin Elmer, NEL542001EA), 200 μ  M GTP (NEB, N0450S)).The run-on reaction was stopped by adding Trizol LS (Life Technologies, 10296-010) and RNA was pelleted with the addition of GlycoBlue (Ambion, AM9515) to visualize the RNA. RNA pellet was resuspended in diethylpyrocarbonate (DEPC)-treated water. RNA was heat denatured at 65 C for 40 s to remove secondary structure. RNA was fragmented using base hydrolysis (0.2N NaOH on ice for 4 min). RNA was purified using streptavidin beads (NEB, S1421S) and removed from beads using Trizol (Life Technologies, 15596-026). We ligated a 3′ adapter ligation using T4 RNA Ligase 1 (NEB, M0204L). We performed a second bead binding followed by a 5′ decapping with RppH (NEB, M0356S). RNA was phosphorylated on the 5' end using T4 polynucleotide kinase (NEB, M0201L) then ligated onto a 5' adapter. A third bead binding was then performed. The RNA was then reverse transcribed using Superscript III Reverse Transcriptase (Life Technologies, 18080-044) and amplified using Q5 High-Fidelity DNA Polymerase (NEB, M0491L) to generate the ChRO-seq libraries. Libraries were sequenced using an Illumina HiSeq by Novogene.