Illumina NovaSeq 6000 paired end sequencing; ChIP-Seq analysis of Myc-tagged budding yeast Rif1
Cultures were grown in YPD media at 30ºC to early exponential phase (0.3 OD/ml at 600 nm) before adding α-factor to a final concentration of 300nM for bar1∆ cells and 3 µM for bar1+ cells. Following the 2-hr incubation, cells were checked under microscopy to ensure that virtually all cells were arrested in G1 phase. To release the cells in S-phase under HU-treatment conditions, cells were collected by centrifugation and resuspended in fresh YPD media containing 200 mM HU and 20 mg/ml Pronase and grown for an additional 75 min at 23ºC.  Formaldehyde was added to a final concentration of 1% formaldehyde and incubated on a gentle tube rotator for 20 min at RT and then, overnight at 4ºC. Finally, 50 OD600 units of cells were harvested by centrifugation and snap frozen in liquid nitrogen and stored at -70ºC until use. Preparation of cleared lysate To prepare the cell lysate, cells were allowed to thaw on ice, before being resuspended in 600 µl lysis buffer (50mM Hepes-KOH pH 7.5, 140mM NaCl, 1mM EDTA, 1% Triton-X100, 0.1% Na-deoxycholate) supplemented with 1x cOmplete protease inhibitor cocktail (Roche, 05892791001) and 1 mM PMSF. Acid-washed glass beads (Merck, G8772) were added to the meniscus, and cells were disrupted using a bead beater (MP Biomedical FastPrep-24) with 5 cycles of ON (6 m/s for 40 s) and OFF (2 min ice). Cell lysates were transferred to a clean 1.5ml Eppendorf tube and disruption of cells was confirmed under a microscope. The sample was then sonicated using a Bioruptor Pico (Diagenode, B01080010) device set on Easy Mode with 6 cycles of 30 sec on and 30 sec. The lysate was then spun at 21,130 g for 30 min at 4ºC, and the supernatant was collected as the cleared lysate.  For each culture condition, 15 µl of cleared lysate was collected as 'Input' and stored at -20ºC.  The remaining cleared lysate was used for the Immunoprecipitation.  Beads-antibody coupling For each immunoprecipitation sample, 25 µl of Dynabeads Protein G (Invitrogen 10004D) suspension was aliquoted and washed in PBS buffer containing 5 mg/ml BSA.  Beads were mixed with 6 µg of anti-Myc tag antibody (Abcam ab9106) and incubated for 10 min at RT and then for at least 4 hours at 4 ºC on a rotator. Finally, beads were washed three times in lysis buffer, resuspended in 100µl of lysis buffer and stored on ice until use.  Immunoprecipitation For each sample to be immunoprecipitated, the antibody-coupled Dynabeads were incubated with 600 µl of cleared lysate overnight at 4ºC on a tube rotator. The following day, beads were washed once with ice-cold lysis buffer, three times with 1 ml ice-cold lysis buffer + 210 mM NaCl, twice with ice-cold wash buffer, once with Ice-cold TE (pH 8.0), and transferred to a fresh tube. The DNA was eluted by adding 50 µl of ChIP elution buffer (50 mM Tris-HCl pH8.0, 10 mM EDTA, 1% SDS) and incubating at 65ºC for 15 min with shaking at 750 rpm. The supernatant was transferred to a fresh tube, and the volume of both input and IP samples was adjusted to 100ul by adding fresh TE + 1% SDS. Both IP and input samples were incubated overnight with shaking at 750 rpm at 65ºC to reverse the crosslinks.  The following day, samples were supplemented with 20 ug of RNase A and were incubated at 37ºC for 1 hr with shaking at 750 rpm. Subsequently, Proteinase K was added to a final concentration of 50 mg/ml and samples were further incubated at 37ºC for 2 hr. Finally, the DNA was purified using a DNA clean and concentrator kit (ZYMO RESEARCH, D5205) and eluted in a 50 µl volume.  The DNA concentration was measured using the Qubit fluorometer (Invitrogen, Q32857). Library DNA was prepared using NEBNext Ultra II DNA Library Prep Kit for Illumina (NEB E7645S) with NEBNext Multiplex Oligos for Illumina (Dual Index Primers Set 1) (NEB E7600S).  Size distribution of the library DNA was checked using TapeStation HS D5000 (Agilent).