title: Illumina HiSeq 4000 sequencing; ChIP-Seq of yeast H3K37me1 in a time-course experiment after release from the G1 cell-cycle phase
design: ChIP-Seq of yeast H3K37me1 in a time-course experiment after release from the G1 cell-cycle phase
library_construction_protocol: 100 mL of yeast cultures per ChIP experiment were collected at the indicated times. Cells were cross-linked with 1% formaldehyde for 15 minutes at room temperature, and the reaction was quenched with 125mM glycine. Cells were resuspended in ChIP SDS buffer (1% SDS, 10 mM EDTA, 50 mM Tris HCl pH 8.0) supplemented with protease inhibitors (Roche) and disrupted with glass beads by using a FastPrep instrument (MP Biomedicals). Yeast strains were grown in YPAD medium at 30C and synchronized to G1 by addition of alpha-factor (10µg/mL final concentration). Chromatin was sonicated (Bioruptor Pico, Diagenode; 9 cycles, 30 s on/off) to yield an average DNA fragment of 300-500 base pairs, and diluted 10 times in ChIP IP buffer (0.01% SDS, 1.1%Triton X-100, 167mM NaCl, 1.2mM EDTA, 16.7mM Tris HCl pH 8.0) supplemented with protease inhibitors (Roche) prior to overnight immunoprecipitation on rotation at 4°C. Next day, 50μl of protein A dynabeads were added, and samples were incubated again on rotation at 4°C for 2 hours. Dynabeads were washed twice with the following buffers: TSE 150 (1% Triton X-100, 0.1%SDS,150 mM NaCl 2 mM EDTA, 20 mM Tris HCl pH 8.0), TSE 500 (1% Triton X-100, 0.1%SDS, 500 mM NaCl 2 mM EDTA, 20 mM Tris HCl pH 8.0) and LiCl buffer (0.25 M LiCl, 1% NP-40, 1% deoxycholate, 1 mM EDTA, 10 mM Tris HCl pH 8.0). DNA was eluted for 30 min at 37°C in elution buffer (100 mM NaHCO3, 1% SDS), and cross-linking was reverted by overnight incubation at 65°C. Samples were treated with 0.5 mg/ml of RNase A at 37°C for 2 h. DNA was purified using the ChIP DNA Clean & Concentrator kit (Zymo Research). Relative DNA amounts were determined by qPCR using Fast SYBR Green Master Mix (Applied Biosystems). Library preparations followed the NEXTflex Rapid DNA-Seq protocol (2015; Bioo Scientific) with a few adjustments. Illumina adaptors were substituted with Bioo NEXTflex adaptors. Agilent Technologies High Sensitivity D1000 was used for determining the fragment size on the Agilent 4200 TapeStation. Libraries were quantified using Invitrogen Qubit dsDNA HS Assay. Equal molarity of each barcoded library was pulled together to multiplex.