Instrument: Illumina HiSeq 2500
Strategy: ChIP-Seq
Source: GENOMIC
Selection: ChIP
Layout: PAIRED
Construction protocol: Cells were fixed with 1% formaldehyde (Sigma-Aldrich) for 15 min at room temperature with mild shaking [120-150 rpm]. The fixing reaction was stopped with 250 mM glycine (RPI) for 15 min at room temperature with mild shaking [120-150 rpm]. Cells were centrifuged and washed once with PBS. The cell pellet was resuspended in lysis buffer [50mM Hepes pH 7.5, 140mM NaCl, 1mM EDTA, 1% Triton X-100, 0.1% Sodium deoxycholate, 1mM PMSF, 1x protease inhibitor cocktail (Roche Applied Science, Penzberg, Germany). Cells were then lysed with 1 ml glass beads (Scientific Industries Inc, Bohemia, NY) at 4oC for 10 min. Both cell lysate and debris were collected and subsequently transferred to an AFA fiber pre-slit snap-cap [6 x 15mm] microtube (Covaris, Woburn, MA) for additional cellular lysis and DNA shearing. Genomic DNA was sheared with E220 focused-ultrasonicator (Covaris) [peak incident power: 75 W, duty factor: 10%, cycles per burst: 200, treatment time: 16 min, temperature: 10oC max, sample volume: 130 µl.] The sheared sample was centrifuged and the clear lysate was collected. Upc2A was immunoprecipitated with Dyna beads-protein G magnetic beads (Invitrogen, Carlsbad, CA) and anti-HA antibody (Invitrogen) [1:50 dilution] overnight at 4oC. Beads were then washed twice with lysis buffer, once with lysis buffer+ 500mM NaCl, once with LiCl buffer [10 mM Tris pH 8, 250 mM LiCl, 0.5% P-40, 0.5% Sodiumdeoxycholate, and 1 mM EDTA], and once with Tris-EDTA buffer. Beads were resuspended in TE and treated with RNAse A (Thermo Fisher Scientific) at 37oC for 30 min. Beads were then washed once with Tris-EDTA buffer, resuspended in Tris-EDTA buffer with 1% SDS, and incubate at 65oC for at least 5 hours to reverse crosslink. Eluted DNA was subsequently purified with mini elite clean-up kit (Qiagen). Qubit fluorometric assay (Thermo Fisher Scientific) was used to analyze the yield quantity and Agilent Bioanalyzer (Agilent Technology, Santa Clara, CA) was used to determine the average sheared DNA size. All ChIPed DNA libraries were generated with Accel-NGS plus DNA library kit (Swift Biosciences, Ann Arbor, MI) according to the manufacturer's instructions.