title: GSM3061432: RSC-ChIP-CGR8-H3K27me3; Mus musculus; ChIP-Seq
library_construction_protocol: Confluent cultures were fixed in PBS with 1% formaldehyde (Sigma) for 8 minutes and quenched using 100 mM fresh glycine for 15 minutes at room temperature. Fixed cell preparations were harvested by scraping, then washed and sonicated in 1% SDS, 30 mM Tris pH 8.0, 150 mM NaCl using a Diagenode Bioruptor UCD-300 to a size range with a median of 200 base pairs. 1 million cell equivalent chromatin was diluted 7x to 15 mM Tris pH 8.0, 1 mM EDTA, 150 mM NaCl, 1% Triton X-100 with fresh protease inhibitor cocktail (Roche) and 0.5 µg H3K27me3 or 1 µg H3K4me3 antibody (Diagenode) and incubated overnight at 4°C with rotation. Protein A/G magnetic beads were washed in the same dilution buffer with 0.15% SDS and 0.1% BSA, added to the chromatin/antibody mix and rotated for 60 min at 4°C. Beads were washed with 400 μl buffer for 5 min at 4°C using (i) first a 150 mM NaCl wash buffer; (ii) then twice a 500 mM NaCl buffer; and (iii) finally a 10 mM Tris buffer. DNA was eluted from the beads using 150 mM NaCl, 30 mM Tris, 1% SDS, 0.1 µg/µl Proteinase K buffer for 30 minutes at 55°C followed by 2 hours shaking at 65°C. The beads were removed after which the DNA was purified using QIAGEN Qiaquick MinElute PCR purification Kit according to manufacturer's protocol and eluted in 20 μl EB. Illumina library preparation was performed using the Kapa Hyper Prep Kit using 5 ng of ChIPped DNA: For end repair and A-tailing double stranded DNA was incubated with end repair and A-tailing buffer and enzyme and incubated first for 30 min at 20°C and then for 30 min at 65°C. Adapters were ligated by adding 30 μl ligation buffer, 10ul Kapa DNA ligase, 5 μl 600 nM adaptor in a total volume of 110 μl and incubated for 15 min at 15°C. Post-ligation cleanup was performed using Agencourt AMPure XP reagent and products were eluted in 20 μl elution buffer. Libraries were amplified by adding 25 μl 2x KAPA HiFi Hotstart ReadyMix and 5 μl 10x Library Amplification Primer Mix using 10 PCR cycles. Samples were purified using the QIAquick MinElute PCR purification kit. Libraries were size selected to ~300bp size using Agencourt Ampure XP beads. Correct size selection was confirmed by Agilent BioAnalyzer. Sequencing was performed using Illumina NextSeq500 with 42bp read lengths.
attributes.tag: GEO Accession
attributes.value: GSM3061432