GSM7728903: ChIPseq_Input_tfb3N-C_Spike_R2; Saccharomyces cerevisiae; ChIP-Seq
ChIP experiments were performed from two independent biological replicates. Yeast cultures were grown in 50 mL of selective medium at 30°C to an OD600 of 0.7-0.9 before crosslinking with 1% formaldehyde (Fisher Scientific, BP531-500) at room temperature for 30 min and quenched with 125 mM glycine. Crosslinked cells were collected by centrifugation and washed twice with 1X TBS (20 mM Tris-HCl pH 7.5, 150 mM NaCl). Cell pellets were then resuspended in 700 μL Lysis buffer (50 mM HEPES-KOH pH 7.5, 140 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% Na-deoxycholate and protease inhibitor cocktail (1 mM PMSF, 1 mM Benzamidine, 10 μg/mL Aprotinin, 1 μg/mL Leupeptin, 1 μg/mL Pepstatin)). About the same number of OD600 units was used for all samples. A fixed amount of crosslinked S. pombe cells (gift from Jason Tanny), representing 10% of the S. cerevisiae cells, were added to the S. cerevisiae cells before cell lysis for normalization, as described before (PMID: 31365865). Cells were lysed by bead beating and the lysate was sonicated with a Model 100 Sonic dismembrator equipped with a microprobe (Fisher Scientific), 4 x 20 sec at output 7 Watts, with a 1 min break between sonication cycles. Soluble fragmented chromatin was recovered by centrifugation. 600 μL of the chromatin sample was taken per immunoprecipitation (IP) and 6 μL (1%) was saved as an Input sample. The following amounts of antibody per IP were used: anti-RNApII Rpb1 8WG16 (BioLegend, 664912; 2 μg), anti-Ser5P 3E8 (gift from Dirk Eick, Chapman et al. 2007 (PMID: 18079404); 5 μL), anti-Tfb1 (Matsui et al. 1995 (PMID: 7708491); 4 μL) and anti-Kin28 (Matsui et al. 1995 (PMID: 7708491); 4 μL). All antibodies have been validated for ChIP. Anti-Ser5P 3E8, anti-Tfb1 and anti-Kin28 antibodies were coupled to Dynabeads coated with Protein G (Thermo Fisher Scientific, 10004D) and anti-RNApII 8WG16 antibody was coupled to Dynabeads coated with Pan Mouse IgG antibodies (Thermo Fisher Scientific, 11042). 50 μL of the appropriate Dynabeads pre-coupled with the indicated antibody were added to the chromatin sample and incubated overnight at 4°C. Beads were washed twice with Lysis buffer, twice with Lysis buffer 500 (Lysis buffer + 360 mM NaCl), twice with Wash buffer (10 mM Tris-HCl pH 8.0, 250 mM LiCl, 0.5% NP40, 0.5% Na-deoxycholate, 1 mM EDTA) and once with TE (10 mM Tris-HCl pH 8.0, 1 mM EDTA). Input and immunoprecipitated chromatin were eluted and reverse-crosslinked with 50 μL TE/SDS (TE + 1% SDS) by incubating overnight at 65°C. Samples were treated with RNase A (345 μL TE, 3 μL 10 mg/mL RNAse A (Sigma-Aldrich, R6513), 2 μL 20 mg/mL Glycogen (Roche, 10901393001)) at 37°C for 2 hr and subsequently subjected to Proteinase K (15 μL 10% SDS, 7.5 μL 20 mg/mL Proteinase K (Thermo Fisher Scientific, 25530049)) digestion at 37°C for 2 hr. Samples were extracted twice with phenol/chloroform/isoamyl alcohol (25:24:1), followed by precipitation with 200 mM NaCl and 70% ethanol. Precipitated DNA was resuspended in 50 μL of TE before being used in ChIP-seq libraries. All ChIP and Input samples were subjected to a 1.7X cleanup using KAPA Pure Beads (Roche, 07983280001) according to the manufacturers' instructions. Samples were eluted with 40 μL of Elution buffer (10 mM Tris pH 8.0). DNA concentration of ChIP and Input samples was determined by qPCR using a standard curve made with a fragment size control sample. 1-10 ng of ChIP and Input DNA were used for ChIP-seq library preparation as follow. The ends of DNA were repaired by incubating in 70 μL of 1X NEBuffer 2 containing 0.6 units of T4 DNA polymerase, 2 units of T4 polynucleotide kinase (NEB, M0201S), 0.09 nM dNTPs and 0.045 μg/μL of BSA at 12°C for 30 min. Repaired DNA was then subjected to a 1.7X cleanup using KAPA Pure Beads before dA tailing as follow. Beads containing the repaired DNA were resuspended in 50 μL of 1X NEBuffer 2 containing 0.1 mM dATP and 25 units of Klenow Fragment (3'→5' exo-) (NEB, M0212M), and incubated at 37°C for 30 min. After a 1.8X cleanup with KAPA Pure Beads, the A-tailed DNA was ligated to index adapters (Illumina Truseq DNA UD Indexes (20023784)) as follow. The beads were resuspended in 45 μL of 1X Ligase buffer containing 8 nM of adapter and 2.5 units of T4 DNA ligase and incubated at room temperature for 60 min. The ligated DNA was then subjected to a 1X cleanup with KAPA Pure Beads, followed by a double size selection (0.52X-1X) leading to fragments in the 200-600 bp range. Libraries were PCR-amplified with 12-13 cycles using KOD Hot Start DNA polymerase (Millipore, 71086-3) and cleaned up using 1X KAPA Pure Beads. Libraries were qualified on Agilent 2100 Bioanalyzer using High Sensitivity DNA Kit and quantified by qPCR using NEBNext Library Quant Kit for Illumina (NEB, E7630). Equal molarity of each library was pooled and subjected to sequencing on Illumina NovaSeq 6000 platform at the McGill University and Génome Québec Innovation Centre to generate 100 bp paired-end reads.