GSM3610434: Rgt1 saturated IP 3; Saccharomyces cerevisiae; ChIP-Seq
Sample information curated by ChIP-Atlas
Antigen
Antigen Class
TFs and others
Antigen
RGT1
Cell type
Cell type Class
Unclassified
Cell type
Unclassified
NA
NA
Attributes by original data submitter
Sample
source_name
TAP-tagged Rgt1
growth conditions
saturated in YPD
Sequenced DNA Library
library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
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) 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.