GSM3333224
GSM3333224: Rpb1_ChIP_wt_t60; Saccharomyces cerevisiae; ChIP-Seq
TAP and RNAPII ChIP-seq was performed as described previously (Ribaud et al., 2012) (PMID: 21952045). Chromatin for MNase digestion was prepared essentially as described in Kubik et al., 2015 (GSE73337) (PMID: 26545077).Cells were pelleted at 3,000 x g and washed three times with 1 mL Buffer A (15 mM Tris pH 7.5, 80 mM KCl, 0.1 mM EGTA, 0.2 mM spermine, 0.5 mM spermidine, Roche cOmplete EDTA-free mini protease inhibitors, 1 mM PMSF), centrifuging as above between washes. Cells were resuspended in 600 µL Buffer A containing 0.1% digitonin and permeabilized at 30C for 5 min. CaCl2 was added to 2 mM and ChEC digests were performed at 30C. 5 min after CaCl2 addition, a 100 µL aliquot of the digest was transferred to a tube containing 100 µL 2X stop buffer (400 mM NaCl, 20 mM EDTA, 4 mM EGTA) and 1% SDS. Protein was then was digested with 80 µg proteinase K at 55C for 30 m. Nucleic acids were extracted with an equal volume of phenol/chloroform/isoamyl alcohol (25:24:1) and precipitated with 2.5 volumes 100% ethanol and 30 µg Glycogen (Roche). Pellets were washed once with 1 mL 100% ethanol, dried, and resuspended in 30 µL 0.1X TE buffer, pH 8.0. RNA was digested with 10 µg RNase A at 37C for 20 m. Prior to generating sequencing libraries, ChEC DNA was subjected to size-selection using Ampure XP beads (ThermoFischer). A beads:sample ratio of 2.5:1 was used and the unbound fraction, containing small DNA fragments, was extracted to remove RNase and precipitated as above, and used for library preparation. TruSeq ChIP library preparation kit (Illumina)
GEO
GSM3333224
GEO Accession
GSM3333224