title: GSM7100833: CN,TOP2Bcc-seq_Input_1; Mus musculus; ChIP-Seq
library_construction_protocol: TOP2Bccs were isolated from sonicated lysates containing covalently bound proteins via native TOP2B ChIP. Prior to immunoprecipitation, 1% of each sample transferred to a clean tube to be used for input normalization. Samples were then incubated with anti-TOP2B antibody (1:50, H-8, sc-25330, RRID:AB_628384) overnight at 4°C followed by incubation with ChIP-grade protein A/G magnetic beads (Thermo) for 3 hours at 4°C. Antibody-conjugated beads were washed with low salt buffer (20 mM Tris-HCl pH 8, 1% Triton-X-100, 2 mM EDTA, 0.1% SDS, 150 mM NaCl), high salt buffer (20 mM Tris-HCl pH 8, 1% Triton-X-100, 2 mM EDTA, 0.1% SDS, 500 mM NaCl), LiCl buffer (10 mM Tris-HCl pH 8, 1% IGEPAL-CA630, 1% deoxycholic acid (sodium salt), 1 mM EDTA, 0.25 M LiCl), and twice with TE buffer (50 mM Tris-HCl pH 8, 1 mM EDTA) for 5 minutes each with agitation at room temperature. Immunoprecipitated TOP2B-DNA complexes were eluted from the magnetic beads in 500 µl ChIP elution buffer (1% SDS, 0.1 M NaHCO3) and purified along with input DNA via phenol-chloroform extraction and ethanol precipitation. Precipitated DNA was washed with 70% ethanol and resuspended in TE buffer. To prepare libraries for sequencing, immunoprecipitated DNA was first end-repaired, A-tailed, and ligated with paired-end, Illumina barcoded adapters (KAPA biosciences) according to the manufacturer's instructions. Attempts to amplify libraries for sequencing after this step alone proved unsuccessful, implying the successful isolation of TOP2Bccs. Following the first round of adapter ligation, the 5'strand was digested with l exonuclease (NEB). The resulting 3'strand was used as a temple for 10 rounds of primer extension (cycling conditions: 95°C for 3 min, ten rounds of linear amplification (95°C for 1 min, 57°C for 1 min and 72 °C for 3 min), 95°C for 1 min, hold at 4°C) using primers against the ligated adapter in a solution containing 1X HiFi Phusion buffer (NEB), 0.8 mM dNTPs, 2 U Phusion DNA polymerase (NEB) and 40 nM of P7 extension primer (CAAGCAGAAGACGGCATACGA*G; *denotes phosphorothioate linkage). The resulting single-stranded products were purified using 0.8X Ampure XP beads (Beckman), eluted in 35 µl TE dH2O, and concentrated to a volume of 18 µl using a vacuum centrifuge (Eppendorf). The purified products were appended with ribo-G in 1X terminal deoxynucleotidyl transferase buffer (NEB), 20 U terminal deoxynucleotidyl transferase enzyme (NEB), and 4 mM of rGTP at 37 °C for 15 min followed by 0.8X Ampure XP bead clean-up and elution as before. The single-stranded, ribo-tailed products were ligated to double-stranded adapters with CCC overhang (oligo 1, AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTCCC; oligo 2, (Phosphate)GATCGGAAGAGCGGTTCAGCAGGAATGCCGAG) as described previously2. Ligated products were used as templates for standard paired-end library amplification (KAPA biosciences) with an additional extension step prior to linear amplification to create double-stranded DNA templates (cycling conditions: 60°C for 3 min, 98°C for 30 sec, eleven cycles of amplification (98°C for 10 s, 65°C for 30 s, and 60°C for 30 s), 60°C for 5 min and hold at 4°C). The final PCR products were purified using 0.8X Ampure XP beads, eluted in 40 µl EB (Qiagen). DNA fragment size was assessed via Tapestation (Agilent) and additional Ampure XP bead clean-up steps were performed to obtain a final library size of 350-450 bp. DNA concentration was quantified by fluorometry (dsDNA high sensitivity Qubit assay, Thermo) to ensure equal DNA concentration for each replicate before replicates were pooled and sequenced on an Illumina NextSeq 500 instrument using V2.5 reagents to an average depth of 25-35 million reads per replicate.