ChIP-seq of Pol III and H3K4me3 in HAP1 and HepG2 tRNA gene deletion clones and control clones
ChIP-seq of Pol III and H3K4me3 in HAP1 and HepG2 tRNA gene deletion clones and control clones
Adherent cells (HAP1 and HepG2) were cross-linked by adding 1% formaldehyde in solution A (50 mM Hepes KOH, pH 7.5; 100 mM NaCl; 1 mM EDTA; 0.5 mM EGTA) for 10 min at room temperature. To stop the cross-linking reaction, 1ml of 2.5 M glycine was added and incubated for 5 min. Cells were wash twice with ice-old 1xPBS and subsequently harvested by using a cell scraper. Cells were pelleted with centrifugation (3.000 xg, 5 min, 4 oC) and washed once with 1xPBS containing protease inhibitors. Cell pellets were frozen in liquid nitrogen and kept at -80 oC. Cross-linked pellets were resuspended in lysis buffer 1 (50 mM Hepes-KOH, pH 7.5; 140 mM NaCl; 1mM EDTA; 10% Glycerol; 0.5% NP-40; 0.25% Triton X-100) for 10 min at 4 oC and pelleted by centrifugation (3.000 xg, 5 min, 4 oC). Pellets were then resuspended in lysis buffer 2 (10 mM Tris-HCL, pH8.0; 200 mM NaCl; 1 mM EDTA; 0.5 mM EGTA) for 5 min at 4 oC and pelleted by centrifugation (3.000 xg, 5 min, 4 oC). The nuclei pellets were resuspended in sonication buffer (10 mM Tris-HCl, pH 8; 1 mM EDTA; 0.1% SDS). Chromatin was sonicated with a Covaris ME220 to a size of 200 bp to 500 bp.  After sonication, 1/50 of the material was taken out and used as input, and the rest of the chromatin material was used for immunoprecipitation. Briefly, the antibody (Pol III or H3K4me3) was coupled to IgG dynabeads overnight at 4 oC. Afterward, the sonicated chromatin material was incubated with the antibody-bead mixture overnight at 4 oC. The ChIP-enriched DNA was eluted from the beads and purified. ChIP-enriched DNA and input control were used as input material for library preparation using Takara SMARTer ThruPLEX DNA-seq Kit according to the manufacture's protocol. DNA samples were first end-repaired during the template preparation step (step 1), followed by adapter ligation during the library synthesis (step 2). Sample indexes were added subsequently, and the libraries were further amplified to achieve enough material for library preparation (step 3). The quality and the size distribution of libraries were determined on an Agilent Bioanalyzer (DNA high-sensitivity chip) instrument according to the manufacturer's protocol. Libraries were quantified with the KAPA-SYBR FAST qPCR kit (Roche).