GSM8008951: GR_pred_1; Mus musculus; ChIP-Seq
Atria- and blood-deprived hearts were cryopowdered using a liquid nitrogen-cooled Retsch Cryomill. The cryopow-dered tissue was then fixed in 10 ml 1% PFA for 30 minutes at room temperature with gentle nutation. Fixation was quenched 1ml of 1.375M glycine (Cat # BP381-5, Fisher Scientific, Waltham, MA) with gentle nutation for 5 minutes at room temperature. After centrifugation at 3000g for 5 minutes at 4°C, the pellet was resuspended in cell lysis buffer as per reported conditions 69, supplementing the cell lysis buffer with 3mg/ml cytochalasin B and rotating for 10 minutes at 4°C.  Nuclei were pelleted at 300g for 10 min at 4°C, and subsequently processed following the reported protocol with the adjustment of adding 3µg/ml cytochalasin B into all solutions for chromatin preparation and sonication, antibody incubation, and wash steps.  Chromatin was then sonicated for 15 cycles (30 sec, high power; 30 sec pause; 500µl volume) in a water bath sonicator set at 4°C (Bioruptor 300; Dia-genode, Denville, NJ).  After centrifuging at 10000g for 10 minutes at 4°C, sheared chromatin was checked on agarose gel for a shear band comprised between ~150 and ~600bp. Two µg of chromatin was kept for pooled input controls, whereas ~50µg chromatin were used for each pull-down reaction in a final volume of 2ml rotating at 4°C overnight. Primary antibody: rabbit polyclonal anti-GR (ABclonal #A2164). Chromatin complexes were precipitated with 100µl Dynabeads M-280 (sheep anti-rabbit #11203D; Thermo Scientific, Waltham, MA).  After washes and elution, samples were treated with proteinase K (cat #19131; Qiagen, Hilden, Germany) at 55°C and cross-linking was reversed through overnight incubation at 65°C. DNA was purified using the MinElute puri-fication kit (cat #28004; Qiagen, Hilden, Germany), quantitated using Qubit reader and reagents. Library preparation and sequencing were conducted at the CCHMC DNA sequencing Core. Up to 30 ng of post-Immunoprecipitation dsDNA as measured by Invitrogen™ Qubit™ high- sensitivity spectroﬂuorometric measurement was made into Illumina-compatible NGS libraries using the ThruPLEX® DNA-Seq kit from Takara Bio Inc. Each created library was dual-indexed with 8-base molecular barcodes for high level mul5plexing. Libraries were made with 13 cycles of PCR ampliﬁcation.