GSM6705547: ChIPseq_TSC1_WT_EED_Input_Rep2; Mus musculus; ChIP-Seq
10 million cells were crosslinked in a 1% formaldehyde solution for 5 minutes at room temperature, after which glycine was added to a final concentration of 125 mM and incubated for 5 minutes to quench the reaction. These fixed cells were centrifuged at 2,500 g for 5 minutes at 4 °C and the pellet was washed twice with 1 mL PBS. Nuclei were extracted by incubating the fixed cells with 500 µL of cell lysis buffer (20 mM Tris-HCl pH 8.0, 85 mM KCl, 0.5% NP40) for 10 min on ice then spun down for 3 min at 2,500 g. The pellet was resuspended in nuclei lysis buffer (10 mM Tris pH7.5, 1% IGEPAL, 0.5% sodium deoxycholate, 0.1% SDS) then sonicated on a Covaris E220 Evolution sonicator (peak incident power 140.0, duty factor 5.0, cycles per burst 200, 20 min). After sonication, chromatin was spun down at 21,000 g for 10 min to pellet insoluble material. The supernatant was transferred to a fresh tube, the volume was increased to 1 ml with chip dilution buffer (0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA, 16.7 mM Tris-HCl pH 8.1, 167 mM NaCl), and 5 µg of EED antibody was added (Abcam ab240650). Immunoprecipitation was carried out at 4 ºC overnight with gentle rotation followed by incubation with protein A dynabeads (Thermo Scientific) for 4 hours the next day. This was followed by two washes of each of the following: low salt wash buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl pH 8.1, 150 mM NaCl); high salt wash buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris, pH 8.1, 500 mM NaCl); LiCl wash buffer (0.25 M LiCl, 1% NP40, 1% deoxycholate, 1 mM EDTA, 10 mM Tris-HCl pH 8.1) and TE buffer pH 8.0 (10 mM Tris-HCl, pH 8.0, 1 mM EDTA pH 8.0). DNA was eluted twice using 50 µl of elution buffer (0.5–1% SDS and 0.1 M NaHCO3) at 65 °C for 15 min. A 16-µl volume of reverse crosslinking salt mixture (250 mM Tris-HCl, pH 6.5, 62.5 mM EDTA pH 8.0, 1.25 M NaCl, 5 mg ml−1 Proteinase K) was added, and samples were allowed to incubate at 65 °C overnight.  The next day 284 µl TE was added to the reaction along with 400 µl phenol-chloroform-isoamyl alcohol (Invitrogen), the solution was briefly vortexed, and then centrifuged at 21,000 g for 10 minutes at room temperature in phase lock tubes (VWR). After centrifugation, the aqueous phase was collected and combined with 20 µL 5M NaCl, 1 µL 20 mg/mL glycogen (Thermo Scientific), and 880 µL 100% ethanol. This solution was stored at -20 ºC overnight and then centrifuged at 21,000 g 4 ºC for 1 hour the next day. This was followed by two washes with 70% ethanol and elution of the pelleted DNA in 50 µL 1x TE. Libraries were prepared using NEBNext Ultra II DNA Library Prep Kit for Illumina (NEB) following the manufacturer's protocol. All libraries were sequenced using 100 bp paired-end sequencing (200 cycles kit) on a NovaSeq 6000 platform.