title: GSM9343462: FKBP-KAP1 A4F3 ES, +dTAG, H3K9me3; Mus musculus; ChIP-Seq
library_construction_protocol: Calibrated native ChIP-seq (cChIP-seq) was performed for H3K9me3 by collecting 5x107 mESCs or NPCs and spiking in 1 × 107 Drosophila Sg4 cells counted using a Countess II FL Automated Cell Counter (Life Technologies). Cells were pelleted by centrifugation and resuspended in 1mL of cold RSB (10mM Tris pH 8.0, 10mM NaCl, 3mM MgCl2) and lysed by addition of 14mL of cold RSB supplemented with 0.1% NP40 and gentle inversion. Nuclei were recovered by centrifugation at 1500 x g for 5 min at 4oC and washed in 4ml RSB with 0.25M sucrose and 3mM CaCl2 supplemented with protease inhibitors. Nuclei were recovered by centrifugation at 1500 x g for 5 min at 4oC and resuspended in 1mL of RSB with 0.25M sucrose and 3mM CaCl2 supplemented with protease inhibitors, which was transferred to a 1.5mL tube for MNase digestion (200U, Fermentas EN0181) of chromatin to mono- and di-nucleosomes at 37oC for 5 min with inversion every minute. The reaction was quenched by addition of 8µL 0.5M EDTA and spun at 5,000rpm for 5min at 4oC. The supernatant (S1) was collected into a new 1.5mL tube and kept on ice. The pellet was thoroughly resuspended in 300µL of Nucleosome Release Buffer (10mM Tris pH 7.5, 10mM NaCl, 0.2mM EDTA) supplemented with protease inhibitors, and incubated for 1hour at 4oC with rotation. This suspension was passed five times through a 27G needle using a 1mL syringe and then spun at 5000rpm for 5min at 4oC. The supernatant (S2) was collected and combined with S1. The chromatin was centrifuged at 13000rpm for 5min to remove any precipitant, and supernatant was collected as chromatin, snap frozen and stored at -80oC until further processing. For immunoprecipitation (IP), chromatin was thawed on ice and 110µL were diluted in 1mL of native ChIP incubation buffer (70mM NaCl, 10mM Tris pH 7.5, 2mM MgCl2, 2mM EDTA, 0.1% Triton X-100) freshly supplemented with protease inhibitors.  100µL were transferred to a new 1.5mL tube and stored at 4oC to be used as input. The remaining diluted chromatin was incubated overnight with rotation with 5µg of H3K9me3 antibody (Abcam, ab8898) or H3K9me3 (Active Motif; AB2532132 Clone 0319; Cat #061013). Samples were then incubated for 90min with 45µL rProtein A Sepharose™ Fast Flow beads (Amersham) pre-blocked with 1mg/mL of yeast tRNA and 1mg/mL BSA. The beads were then washed five times for 10min with rotation at 4oC with Wash Buffer (20mM Tris pH 7.5, 2mM EDTA, 125mM NaCl, 0.1% Triton X-100) followed by one final wash in 1x TE. The DNA was then eluted from the beads by incubation at 25oC for 30 min shaking at 1000rpm with 1% SDS, 100 mM NaHCO3 elution buffer and separated from the beads by centrifugation. DNA from the IP elutions and input samples was further purified using the ChIP DNA Clean and Concentrator Kit (Zymo Research) according to manufacturer's instructions. ChIP and input DNA was quantified using the Qubit High Sensitivity DNA kit (Invitrogen) and submitted to library preparation using the the NEBNext Ultra II DNA Library Prep Kit (New England Biolabs) according to the manufacturer's instructions. Libraries were quantified using the Qubit High Sensitivity DNA kit (Invitrogen) and their length was estimated using the Bioanalyzer High Sensitivity DNA kit (Agilent) before pooling and subjected to paired-end sequencing on an Illumina NextSeq 500 machine using NextSeq 500/550 High-Output v2.5 Kit (150 cycles) (Illumina).