title: GSM2579668: RPC Elute 3; Saccharomyces cerevisiae; ChIP-Seq
library_construction_protocol: For chromatin preparation, cultures were cross-linked for 15 min by addition of 1/10 culture volume of cross-link solution (50 mM HEPES-KOH pH 7.5, 100mM NaCl, 1 mM EDTA pH 8, 11% formaldehyde) at room temperature with very light shaking. Next cultures were quenched with 300 mM glycine and light shaking for 5 min. Cells were pelleted and washed once with cold 1X PBS, decanted, and frozen in dry ice. Cell pellets were resuspended in Extraction Buffer (50 mM HEPES-KOH pH 7.5, 140 mM NaCl, 1 mM EDTA pH 8, 1% Triton-X) plus protease inhibitor cocktail (Sigma-Aldrich). Approximately 0.6 g of glass beads were added and cells were mechanically sheared at 4°C by vortexing for 30 min. Lysates were pelleted at 4°C for 5 min and supernatants cleared. Chromatin pellets were resuspended in 700 µl of Extraction Buffer, divided into two aliquots and sonicated for 7-10 min in a Biorupter Standard (Diagenode). For immunoprecipitations, 20 µl of Anti-FLAG M2 Magnetic Beads (Sigma-Aldrich) were washed once in 1 mL of Extraction Buffer and incubated with 50 µg of chromatin and rotation overnight at 4°C. Following incubation, beads were washed in Extraction Buffer with increasing concentrations of NaCl: 140 mM, 300 mM, 600 mM and final wash with 250 mM LiCl. Each wash was done for 5 minutes at 4°C with rotation. Beads were resuspended in 200 µl of 1X HE (10 mM HEPES-KOH pH 7.5, 1 mM EDTA). For input control samples 5 µgs of chromatin was diluted into 200 µl of 1X HE. IPs and input samples were RNased (20 µg) for 1 h at 37° C followed by Pronase E (40 µgs) treatment for 4-5 hours. DNA was purified using the Zymo DNA Clean and Concentrator kit with 5X ChIP-DNA Binding Buffer (Genesee, Inc.). ChIP-seq libraries were prepared according to the Illumina Multiplex Sample Preparation Guide. Immunopurified DNA or input control DNA samples were treated with the Fast DNA End Repair kit (Thermo Scientific) to creat blunt DNA ends, then adenylated on their 3’-end using the Klenow Exo- enzyme with 1 mM dATP (Fisher Scientific), and ligated to adapters. Individual IP or input samples were ligated to a unique multiplex barcoded adapter set for sequencing. Following ligation, samples were purified using the Agencourt AMPure XP purification beads (Beckman Coulter, Inc.). Nine replicate PCR reactions with ~ 4-6 ngs of DNA template and Illumina universal primers was done using KOD polymerase with conditions 95°C 5 minutes, 5 cycles of 95°C 30 seconds 65°C 30 seconds 70°C 30 seconds, followed by 10 cycles of 95°C 30 seconds 61°C 30 seconds 70°C 30 seconds, and a final 70°C for 2 minutes. All sequencing libraries were first evaluated by gel electrophoresis, then submitted to the University of California, Irvine Genomics High-throughput Facility for Bioanalyzer 2100 (Agilent) analysis, KAPA quantification for sample mixing and paired-end 100 base sequencing on the Illumina Hi-Seq 2500.
attributes.tag: GEO Accession
attributes.value: GSM2579668