GSM4696197: ChIPexo_Rpb3_chd1D_R1; Saccharomyces cerevisiae; ChIP-Seq
ChIP-exo experiments were performed essentially as described previously (Rossi et al., 2018) with some modifications. After following our standard ChIP protocol using a crosslinked 50 mL cell culture at an OD600~0.8 per IP and the appropriate antibody-coupled Dynabeads (see above), beads were washed twice with Lysis buffer, twice with Lysis buffer 500, twice with Wash buffer and once with 10mM Tris-HCl pH8.0. Beads were then briefly centrifuged to remove all liquid before being used in the ChIP-exo library as follows. For A-tailing of the immunoprecipitated DNA, beads were resuspended in 50 μL of 1X NEBuffer 2 (NEB, B7002S) containing 0.1 mM dATP and 15 units of Klenow Fragment (3'→5' exo-) (NEB, M0212M) and incubated at 37˚C for 30 min. After washing the beads with 10 mM Tris-HCl pH 8.0, dA-tailed DNA was ligated to the First adapter by resuspending the beads in 45 μL of 1X Rapid ligation buffer (Enzymatics, B1010) containing 375 nM of First ligation adapter (synthesized by IDT; see Table S2 for a list of adapters with their bar codes), 10 units of T4 polynucleotide kinase (NEB, M0201S) and 1,200 units of Rapid T4 DNA ligase (Enzymatics, L6030-HC-L) and incubating at room temperature for 60 min. After washing the beads with 10 mM Tris-HCl pH 8.0, the ligated DNA was subjected to a Fill-in reaction by resuspending the beads in 40 μL of 1X phi29 buffer (NEB) containing 0.2 mg/mL BSA (NEB), 180 μM dNTPs and 10 units of phi29 DNA polymerase (NEB, M0269S) and incubating at 30˚C for 20 min. After washing the beads with 10 mM Tris-HCl pH 8.0, DNA was subjected to λ exonuclease digestion by incubating the beads with 50 μL of λ exonuclease reaction buffer (NEB) containing 0.1% Triton X-100, 5% DMSO and 10 units of λ exonuclease (NEB, M0262S) at 37˚C for 30 min. To elute and reverse-crosslink DNA, beads were resuspended in 40 uL of reverse crosslink mix containing 25 mM Tris-HCl pH7.5, 200 mM NaCl, 2 mM EDTA, 0.5% SDS and 30 μg of Proteinase K (Thermo Scientific, 25530049) and incubated at 65˚C for 16 hr. Eluted DNA was then subjected to a 1.8X cleanup using KAPA Pure Beads (Roche, 07983280001) according to the manufacturers' instructions before proceeding with Second adapter ligation as follows. Beads were resuspended in 40 μL of 1X T4 DNA ligase reaction buffer (NEB, B0202S) containing 375 nM of Second ligation adapter (synthesized by IDT; see Table S2) and 1,200 units of Rapid T4 DNA ligase (Enzymatics, L6030-HC-L), and incubated at room temperature for 60 min. The ligated DNA was then subjected to a 1.8X cleanup with KAPA Pure Beads and eluted in 15 μL ddH2O. Libraries were PCR-amplified in 40 μL reactions with 15-18 cycles using KOD Hot Start DNA polymerase (Millipore, 71086-3) and 500 nM of each primer P1.3 and P2.1 (synthesized by IDT; see Table S2). At this step, 10 μL from each sample was further amplified for six more cycles to visualize the quality of the library by electrophoresis on a 2% TAE-agarose gel stained with ethidium bromide. Once the library validated, DNA was subjected to a 1X cleanup with KAPA Pure Beads, followed by a double size selection (0.6X-1X) leading to DNA fragments in the 150-450 bp range. Libraries were qualified on Agilent 2100 Bioanalyzer using High Sensitivity DNA kit (Agilent Technologies, 5067-4626) and quantified by qPCR using NEBNext Library Quant kit for Illumina (NEB, E7630). Equal molarity of each library were pooled (14-16 libraries per pool) and subjected to sequencing on an Illumina HiSeq 4000 platform at the McGill University and Génome Québec Innovation Centre to generate 50 bp paired-end reads. Barcode ID #1:  1st ligation adapter (D702) Barcode ID #2:  2nd  ligation adapter (D503) ChIP-exo