title: GSM3610443: Rgt1_exponential_input_3; Saccharomyces cerevisiae; ChIP-Seq
library_construction_protocol: Crosslinked cell pellets were resuspended in 500 µl FA lysis buffer with 1x Pierce EDTA-free protease inhibitor cocktail, and then 700 µl of 500 µm acid-washed glass beads were added. The cells were vortexed for 12 minutes total, in cycles of 2 minutes shaking and 2 minutes resting on ice. The lysate was pelleted and resuspended in fresh lysis buffer, and then sonicated for 3x10 min runs on a Diagenode Bioruptor, on high power with cycles of 30 seconds on and 30 seconds off, to an average of ~300 bp. The sonicate was cleared by centrifugation, and the resulting supernatant was split into an input aliquot (1/20 of IP volume) and an IP aliquot. For each sample, 10 ul of Pan-Mouse IgG beads (Thermo Fisher Scientific catalog #11041) were washed in fresh lysis buffer for 2 hours and then added to the lysate and then incubated overnight. After washing the beads twice in FA lysis buffer, once in lysis buffer with 500 mM NaCl, twice in RIPA buffer, and once in TE, and the DNA was eluted in 100 ul TE+1% SDS and a second time in 150 ul TE+0.67% SDS. The eluate and inputs were treated with RNase A and Proteinase K and then reverse crosslinked overnight at 65C. DNA was purified using Zymo ChIP DNA Clean & Concentrator and eluted in 15 µl 10 mM Tris-HCl pH 8. ChIP-seq libraries were prepared using the Accel-NGS 2S Plus DNA Library Kit (Swift) with dual indexing, from either 1 ng total from input samples or 10 ul of IP samples. Input and IP libraries were amplified for 9 and 12-15 cycles, respectively.
attributes.tag: GEO Accession
attributes.value: GSM3610443