Trac-looping protocol
Keji Zhao, Qingsong Tang, Binbin Lai · Protocol Exchange · 2019
Long-range chromatin interactions play critical roles in genome organization and regulation of transcription.We developed a novel technique, Transposase-mediated analysis of chromatin looping \(Trac-looping), for simultaneous detection of multiscale genome-wide chromatin interactions among regulatory elements and chromatin accessibility.This document describes how to generate Trac-looping library. ReagentsBuffers: Lysis buffer: 50mM Tris-HCl pH8.0, 300 mM NaCl, 20mM Imidazole, 0.1% Triton X-100, 10 μg/ml Pepstatin A \(calbiochem cat# 516481), 10 μg/ml Leupeptin Hemisulfate \(calbiochem cat# 108975), 10 μg/ml Chymostatin \(calbiochem cat# 230790), 6 μg/ml Antipain Dihydrochloride \(Sigma cat#A6191), 1mg/ml lysozyme.Wash buffer: 50mM Tris-HCl pH8.0, 1M NaCl , 20mM Imidazole, 0.1% Triton® X-100.Elution buffer: 50mM Tris-HCl pH8.0, 1M NaCl, 250mM Imidazole, 0.1% Triton® X-100.10x annealing buffer: 0.5 M Tris-acetate pH 7.5, 1.5 M potassium acetate, 40 mM spermidine.RSB buffer: 10mM Tris HCl pH7.4,10mM NaCl, 3mM MgCl2.Tn5 cell lyse buffer: 10mM Tris-HCl pH7.5, 10mM NaCl, 3mM Mg Cl2 and 0.5% NP40.10x Tn5 reaction buffer: 0.5 M Tris-acetate \(pH 7.5), 1.5 M potassium acetate, 100 mM magnesium acetate, 40 mM spermidine.1X T7 DNA Ligase Reaction Buffer: 66 mM Tris-HCl, 10 mM MgCl2, 1 mM ATP, 1 mM DTT, 7.5% Polyethylene glycol \(PEG 6000), pH 7.6 @ 25°C Tn5 Enzymes.The Tnp enzyme is stored at -80°C freezer in elution buffer with nal 1 mM dithiothreitol and 50% glycerol.Oligonucleotides.All oligos are ordered from IDTDNA.com.