Instrument: NextSeq 500
Strategy: ChIP-Seq
Source: GENOMIC
Selection: ChIP
Layout: SINGLE
Construction protocol: For breaking cells, 500 OD600 of cells were thawed and resuspended in 4 ml of FA buffer (50 mM HEPES, pH 7.5; 140 mM NaCl; 0.1% SDS; 1 mM EDTA; 1% Triton X-100; 0.1% Deoxycholate, Na salt) with 1/100 PIC (Protease inhibitor cocktail Set IV, in DMSO; 539136, Merck) and 1 mM phenylmethanesulfonyl fluoride (PMSF, P7626, Sigma). The cell suspension was divided into four 2-ml breaking tubes (Biospec, 10832, Microtube 2 ml with cap) containing 1 ml volume of glass beads (11079105, BioSpec Products). The lysis was performed by three cycles of 5-min beating followed by 1-min chilling on ice (Biospec Mini-BeadBeater-16). Cell lysates and glass beads were separated by punching a hole on the bottom of the tube with a red-hot 18G needle and slow centrifugation (500 g, 3 min, 4°C; Eppendorf 5810R centrifuge, A-4-62 rotor). The collected lysates were combined into a 15-ml centrifuge tube and washed with two cycles of 5 ml FA buffer followed by centrifugation (12K rpm, 5 min, 4C; Eppendorf 5810R centrifuge, F-34-6-38 rotor). The washed lysates were completely resuspended in 2 ml FA buffer with 1/100 PIC and 1 mM PMSF in a new 15-ml centrifuge tube (CFT011150, BIOFIL). Chromatin was sheared by sonication in a Bioruptor water bath sonicator (Diagenode) with 15 ml tube-chip unit (High intensity; 30 sec on, 30 sec off, 15 min/cycle; total 3 cycles) at 4°C. The sheared lysate was centrifuged at 12000 g for 10 min at 4°C (Eppendorf 5810R centrifuge, F-34-6-38 rotor). About 25 ml of supernatant corresponding to 5 OD600 of cells was collected, mixed with 200 ml TES buffer (50 mM Tris-Cl, pH 8; 10 mM EDTA, pH 8; 1% SDS) plus 175 ml TE buffer (10 mM Tris-Cl; 1 mM EDTA, pH 8), and stored at -20°C as the input-DNA. Immunoprecipitation was performed by incubating all the rest sheared chromatins with 300 ml Dynabeads® Pan Mouse IgG (11041, human anti-mouse IgG, Invitrogen) in 6 ml of FA buffer with 1/100 PIC and 1 mM PMSF. The binding was performed in a 15-ml tube with mixing using an end-over-end rotator (Fntelli-Mixer, RM-2L, F1 mode, 30 rpm) at 4°C for 16 h. The magnetic beads were anchored with DynaMagTM-2 Magnet and the bound complexes were washed 4 times with 1 ml of each wash buffer for 5 min [FA buffer × 1, high-salt FA buffer (50 mM HEPES, pH 7.5; 500 mM NaCl; 0.1% SDS; 1 mM EDTA; 1% Triton X-100; 0.1% Deoxycholate, Na salt) × 1, DOC buffer (10 mM Tris·Cl; 1 mM EDTA, pH 8; 25 0mM LiCl; 0.5% IGEPAL® CA-630; 0.5% Deoxycholate, Na salt) × 1, and TE buffer × 1] with mixing (Fntelli-Mixer, RM-2L, F1 mode, 30 rpm) at room temperature. The bound complexes were eluted twice by heating at 65°C in 200 ml TES buffer for 20 min and in 200 ml TE buffer for 10 min. Two eluates were pooled as the ChIP-DNA. To remove RNA, both the input-DNA and ChIP-DNA (~400 ml each) were treated with 5 ml of 10 mg/ml RNase A (R5503, Sigma; 100 mM Tris-Cl, pH 7.4) at 37°C for 30 min. For de-crosslinking, both the RNase-treated input-DNA and ChIP-DNA were treated with 40 ml of 20 mg/ml Proteinase K (1.24568.0500, Merck; in 50 mM Tris-Cl, pH 8) at 42°C for 1 h and then 65°C for 16 h. De-crosslinked DNA was purified using the QIAquick DNA Purification Kit (28106, Qiagen) according to the manufacturer's instruction, with a modification of a two-cycle wash step using the PE buffer. Final ChIP-DNA and input-DNA were eluted with 50 ml EB buffer. DNA concentrations were measured by using the Qubit™ dsDNA HS Assay Kit (Q32854, Invitrogen by Thermo Fisher Scientific). ChIP-seq analysis was performed from three biological replicates of YPD- and YPGly-grown cells. The average fragment length of sonicated fragments was 150–1000 bp. For each condition, libraries were prepared from 3.96–20 ng of ChIP-DNA and input-DNA using the KAPA LTP Library Preparation Kit for Illumina® platforms (KK8232, KAPA Biosystems by Roche) according to the manufacturer's instruction. Notably, after adapter ligation, double-sided size selection between 250–450bp for adapter-ligated fragments was performed using the KAPA Pure Beads (KK8000/07983271001, KAPA Biosystems by Roche). Size-selected fragments were then PCR-amplified for 12 cycles. The size of each library was assessed using a Bioanalyzer 2100 instrument (Agilent Technologies) with the High Sensitivity DNA Kit (Agilent Technologies). The concentration of each library was quantified by using the Qubit™ dsDNA HS Assay Kit (Q32854, Invitrogen by Thermo Fisher Scientific) and qPCR. Single-read sequencing (75 bp) of the libraries was performed using the NextSeq 500/550 high output reagent kit V2_75 cycle (FC-404-2005, Illumina) on an Illumina NextSeq500 sequencer.