alias: GSM4872754
title: GSM4872754: ha-fast1a-asf-async-x16; Saccharomyces cerevisiae; ChIP-Seq
library_construction_protocol: ChIP was carried out as detailed in Gutin et all, Cell reports 2019, with following modifications. Cell pellets (each from 50 ml of cultures at OD600 0.4, 20 ODs total) were thawed on ice and washed in 10 ml 1M sorbitol. After complete liquid removal, pellets were resuspended in 600 ul RIPA buffer (10 mM Tris pH 8.0, 140 mM NaCl, 1 mM EDTA, 0.1% SDS, 0.1% sodium deoxycholate, 1% Triton X-100, EDTA-free protease inhibitor cocktail) and transferred to chilled LoBind Eppendorf microcentrifuge tubes containing ~500 ul of 0.5 mm zirconium oxide beads (Next Advance, ZrOB05). Cells were processed for 3 cycles in a Bullet Blender 24 (Next Advance) at level 8 for 1 min, with 1 minute on ice between cycles. Debris and lysate were transferred by piercing a hole in the bottom of the tube, placing a clean chilled tube under and centrifuging at 600xg for 5 seconds at 4C. The upper tube with the Zirconium beads was discarded and the lysate was hard spun at 17,000xg for 10 minutes at 4C. The supernatant containing cleaved solubilized 8xmyc tags was discarded, and the pellet was thoroughly resuspended in 100 ul NP buffer (10 mM Tris pH 7.4, 1 M sorbitol, 50 mM NaCl, 5 mM MgCl2, 1 mM CaCl2, and 0.075% NP-40, freshly supplemented with 1 mM β-mercaptoethanol, 500 μM spermidine, and EDTA-free protease inhibitor cocktail), and SLIM-ChIP protocol was carried continued as in Gutin et al. SLIM-Chip, as per Gutin et al, Cell Reports 2019
identifiers.namespace: GEO
identifiers.id: GSM4872754
attributes.tag: GEO Accession
attributes.value: GSM4872754