title: GSM3756700: ATAC-seq Nrd1-AA_drpd3_+Rap; Saccharomyces cerevisiae; ATAC-seq
library_construction_protocol: The experiment was performed after combining the protocols from (Buenrostro et al., 2013; Buenrostro et al., 2015; Schep et al., 2015) and (Au - Grbesa et al., 2017). The yeast strains were inoculated the day before in YEPD. Culture was diluted to OD600 of 0.2 and it was allowed to reach an OD600 of 0.4. Then, half of the culture is treated with Rapamycin for 1h at 30°C. 1 mL of the cell culture was harvested from all conditions and strains. Cells were washed twice in 1 mL sorbitol buffer (1.4 M Sorbitol, 40 mM HEPES-KOH, pH 7.5, 0.5 mM; MgCl2) and were resuspended in 190 uL of sorbitol buffer and 10 uL of 10 mg/mL Zymolyase (20T Zymolyase, AMSIO) and incubated at 30 °C shaking at 200 rpm for 30 minutes. The cells were washed twice in sorbitol buffer and then the nuclei were incubated in 95 uL of 1x TD buffer and 5 uL of the TD enzyme (Illumina). The reaction was incubated at 37 °C at 500 rpm for 30 minutes. DNA was then extracted with 3x SPRI beads (AmpureXP Beckman Coulter) and was eluted in 28 uL 10 mM Tris-HCl, pH 8.0. Initial PCR amplification was performed with 4 uL of transposed DNA to check the transpositions and cycles required. Final amplification was performed with NEBNext High-Fidelity 2x PCR Master Mix (NEB) using Nextera Primers (Illumina). 12 cycles were performed.The size selection was performed by sequential SPRI beads precipitation. First, the DNA was mixed by pipetting in 0.5x beads followed by incubation for 5 minutes at room temperature. The supernatant was used for next DNA precipitation with 2.5x SPRI beads after which the supernatant was discarded and the beads were washed with 70% ethanol twice before the DNA was extracted using 28 uL of 10 mM Tris-HCl, pH 8.0.  The DNA concentration was measured and the samples were paired-end sequenced at iGE3 genomics platform of the University of Geneva. NEBnext Ultra DNA library prep kit for Illumina (NEB)
attributes.tag: GEO Accession
attributes.value: GSM3756700