ChIP-seq: Lysates were clarified from sonicated cells and protein-DNA complexes were isolated with antibody or with streptavidin-magnetic beads for biotinylated protein. Libraries were prepared according to Illumina's instructions. Briefly, DNA was end-repaired using a combination of T4 DNA polymerase, E. coli DNA Pol I large fragment (Klenow polymerase) and T4 polynucleotide kinase. The blunt, phosphorylated ends were treated with Klenow fragment (minus exo) and dATP to yield a protruding 3- 'A' base for ligation of Illumina's adapters which have a single 'T' base overhang at the 3’ end. After adapter ligation, ChIP DNA was PCR-amplified with Illumina Genomic adaptors or with NEXTflex DNA barcodes adaptors for 18 cycles and library fragments (ChIP) (insert plus adaptor and PCR primer sequences) were size-selected (150-250bp) from a 2% agarose gel. The purified DNA was captured on an Illumina flow cell for cluster generation. Libraries were sequenced on a Genome Analyzer II (Illumina) or HiSeq 2000 (Illumina) following the manufacturer's protocols. GRO-seq: GRO-Seq was performed as described (Core, L.J., Waterfall, J.J., and Lis, J.T., Science 322, 1845-1848 (2008)) with minor modifications. Approximately 10 million nuclei per sample were extracted and used for run-on and BrU incorporation. BrU labeled nascent transcripts were immunoprecipiated with anti-BrU agarose beads (Santa Cruz Biotech), washed, eluted and precipitated in ethanol. BrU precipitated RNA were subjected for first strand complementary DNA synthesis. First, polyA tailed was added using Poly(A)-polymerase (NEB). Reverse transcription was then performed using Scriptscript III Reverse Transcriptase (Invitrogen) with 5' phosphorylated primer containing an abasic dSpacer furan with binucleotide barcode. Excess primers were removed by Exonuclease I (Fermentas). First-strand cDNA products were fragment with basic hydrolysis and size-selected (105-400 nt) in a 10% polyacrylamide TBE-urea gel (Invitrogen). cDNA was subsequently circularlized using CircLigase (Epicentre), and relinearized at the abasic dSpacer furan with ApeI (NEB). The ssDNA template was amplified to generate DNA for sequencing by Phusion Hig-Fidelity DNA Polymerase (Thermo Science). PCR product was purified and size selected (140-225 bp) by gel electrophoresis on a non-denaturing 8% polyacrylamide TBE gel (Invitrogen). Purified DNA was then sequence on Illumina Genome Analyzer II according to the manuactuere’s instructions. 5'GRO-seq: For each RAW264.7 cell line, 20x106 nuclei were prepared from approximately 30x106 cells. Nuclear run-ons were performed in parallel on 100 μl aliquots containing 5x106 nuclei as for conventional GRO-Seq. Reactions were stopped and RNA was extracted with 450 μl TRIzol LS reagent (Invitrogen) each according to the manufacturer’s instructions. Following DNase treatment, the RNA was hydrolyzed in 20 μl total volume with 2 μl RNA fragmentation buffer (Ambion) for 10 minutes, and divalent cations were removed by gel filtration. Fragmented RNA was then 3’-dephosphorylated with polynucleotide kinase (Enzymatics), for 2 h at 37°C. The reaction was stopped with EDTA and PNK was inactivated and RNA denatured by heating the reaction to 75°C for 5 minutes, then cooled on ice for 2 minutes. BrdU-containing RNA fragments were precipitated using anti-BrdU agarose beads. The resulting RNA was dephosphorylated with calf intestinal phosphatase (NEB) and 5’-de-capped with tobacco acid pyrophosphatase (Epicentre). The reaction was stopped and RNA was extracted with Trizol LS, and libraries were prepared by ligating Illumina TruSeq-compatible adapters to the RNA 3’ and 5’ ends with truncated mutant RNA ligase 2 (K227Q) and RNA ligase 1 (NEB), respectively, followed by reverse transcription, cDNA isolation, and PCR amplification for 12 cycles. Final libraries were size-selected on PAGE/TBE gels to 60-110 bp insert size. A detailed protocol is available on request.