title: GSM2551234: Taf2_YPD_3IAA_1; Saccharomyces cerevisiae; ChIP-Seq
library_construction_protocol: Cells were rapidly harvested by vacuum filtration through a 90 mm diameter 0.45 micron nitrocellulose filter, followed by a 100 ml wash with the cell culture media.  The cell paste was scraped off with a pre-chilled spatula and plunged into liquid nitrogen to flash freeze cells.  For spike-in normalization, an 8 liter batch of S. pombe was grown in YE (+ Antifoam 204) to a similar absorbance and harvested as described above.  2.5 g S. cerevisiae were combined with 0.25 g S. pombe in a mortar pre-chilled on dry ice and liquid nitrogen. Frozen cells were lysed with a 4.5” OD mortar and large pestle. Cells were ground for 1 min, liquid nitrogen was then added to the cells and ground for 1 min.  The grinding and addition of liquid nitrogen was repeated for a total of 6 minutes.  The frozen powdered ground cells were transferred to a cold conical tube and stored at -80 deg. DNA isolated from IPs was prepared for sequencing as previously described (Skene and Henikoff, eLIFE 2017), except for the following changes.  End Repair and A-tailing was performed in a 20 ml volume containing up to 50 ng DNA and the following final concentrations:  1X T4 DNA ligase buffer (NEB #B0202S), 0.5 mM each dNTP, 0.25 mM ATP, 2.5% PEG 4000, 2.5 U T4 PNK (NEB #M0201S), 0.05 U T4 DNA polymerase (Invitrogen #18005025), and 0.05 U Taq DNA polymerase and reactions were incubated at 12 deg 15 min, 37 deg 15 min, 72 deg 20 min, then put on ice and immediately used in adapter ligation reactions.  Adapter Ligation was performed in a 50 ml volume containing 100:1 adapter:insert molar ratios, 1X Rapid DNA ligase buffer (Enzymatics #B101L) and 3000 U DNA ligase (Enzymatics #L6030-HC-L).  A single post-ligation cleanup was performed using 0.4X vol AMPure XP reagent (Beckman Coulter #A63881) and DNA eluted in 40 ul 0.1X TE.  Library Enrichment was performed in a 25 μl volume containing 16.25 μl DNA from previous step and the following final concentrations:  1X KAPA buffer, 0.3 mM each dNTP, 0.5 mM each P5 and P7 PCR primer, and 0.5 U KAPA HS HIFI polymerase (#KK2502).  DNA was amplified with the following program:  98 deg 45 sec, [98 deg 15 sec, ramp to 60 deg @ 1 deg/sec, 60 deg 20 sec, ramp to 98 deg @ 1 deg/sec] 8-10X, 72 deg 1 min.  A post-PCR cleanup was performed using 1.4X vol AMPure XP reagent and DNA was eluted into 40 ml 0.1X TE.  Libraries were sequenced on the Illumina HiSeq2500 platform using 25 bp paired-ends at the Fred Hutchinson Cancer Research Center Genomics Shared Resources facility.
attributes.tag: GEO Accession
attributes.value: GSM2551234