title: GSM9323868: 60min rep2 ChIP-seq; Saccharomyces cerevisiae; ChIP-Seq
library_name: GSM9323868
library_construction_protocol: For chip-seq, Yeast cultures (50 mL) were grown in SCD–Met medium to an OD660 of 0.4. Crosslinking was performed by adding 1.39 mL of 37% formaldehyde directly to each culture and incubating at room temperature for 20 min. Crosslinking was quenched with 2.7 mL of 2.5 M glycine for 5 min. Cells were pelleted at 3000 rpm for 3 min at 4 °C, resuspended in 1 mL TBS, and washed twice with 1 mL TBS. Cells were lysed by resuspension in 250 μL FSPP buffer (50 mM HEPES-KOH pH 7.5, 140 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate, 1% protease inhibitor cocktail, 1 mM PMSF) with 300 μL glass beads. Samples were vortexed at 4 °C twice for 20 min each, separated by a 10 -min break. Lysates were released by piercing the tube ends with a hot needle and centrifuging at 3000 rpm for 5 min at 4 °C into 15 mL tubes. Lysates were recovered, supplemented with 1 mL FSPP buffer, and sonicated (30 s on/off cycles for 7 min). After centrifugation at 14,000 rpm for 20 min at 4 °C, 200 μL supernatant was saved as input control. For immunoprecipitation, 200 μL of lysate was diluted with 800 μL FSPP buffer and incubated with 20 μL pre-blocked magnetic IgG beads and 1–2 μg of antibody (TAP: Thermo CAB1001) overnight at 4 °C with rotation. Beads were sequentially washed with 1) FA Lysis buffer (50 mM HEPES-KOH pH 7.5, 140 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate), 2) FA Lysis + 150 mM NaCl, 3) FA Lysis + 500 mM NaCl, 4) LiCl buffer (0.25 M LiCl, 1% NP-40, 1% sodium deoxycholate, 1 mM EDTA, 10 mM Tris-HCl pH 8.0), and 5) TE buffer (pH 8.0). ChIP sample was then eluted with 400 μL elution buffer (50mM NaCl, 50mM Tris-HCl, pH 8.0, 10mM EDTA, 1% SDS) by rotating at 30°C for 30 min. Eluates were treated with 5 μL of 20 mg/mL proteinase K overnight at 65 °C. Input controls were processed in parallel by adding 160 μL FA Lysis buffer, 40 μL 10% SDS, and 5 μL proteinase K, followed by overnight incubation at 65 °C. DNA was extracted with phenol:chloroform:isoamyl alcohol. ChIP samples were resuspended in 40 μL H20. Input samples were resuspended in 380 μL TE buffer with 10 μL of 10mg/mL RNase A and incubated at 37°C for 30 minutes. DNA was washed with EtOH and resuspended in 40 μL H20. Factor enrichment was verified through qPCR. MNase and ChIP sequencing libraries were prepared using NEBNext ® Ultra™ II DNA Library Prep Kit for Illumina® (Catalog # E7645L) according to the manufacturer's instructions. The sequencing libraries were sequenced on a NextSeq 2000 machine in 2 x 50 bp paired-end sequencing mode at the Genomics Research Incubator at The Pennsylvania State University.
identifiers.namespace: GEO
identifiers.id: GSM9323868_r1