GSM9291275: E14 TKO AEBP2 short rescue ChIP AEBP2 rep1; Mus musculus; ChIP-Seq
CUTnTag: CUT&Tag was carried out following the Epicypher protocol.  ChIP-seq: 3 x 106 cells were harvested and fixed sequentially with 2mM disuccinimidyl glutarate (DSG; Thermo Fisher, 20593) for 35 minutes followed by 1% formaldehyde for 10 minutes at room temperature. Crosslinking was quenched with 0.125M glycine, and nuclei were isolated in NE buffer and extracted nuclei were then resuspended 1% SDS lysis buffer (50mM Tris-HCl pH 8, 10mM EDTA pH 8, 1% SDS) and sonicated by Covaris S220 sonicator (Power 175 W, Duty 10%, 200 cycles per burst) for 430 seconds to generate DNA fragments of approximately 200bp. Sheared chromatin was diluted in ChIP dilution buffer (16.7 mM Tris-HCl, pH 8.0, 0.01% SDS, 1.1% Triton X-100, 16.7 mM NaCl, 1.2 mM EDTA) and clarified by centrifugation at 13,000 rpm for 15 minutes at 4 °C. Chromatin was incubated overnight at 4 °C with Dynabeads protein A pre-bound to 2.4mg target antibody. 1 µg of Drosophila chromatin (Active Motif, #53083) and 0.1 µg of anti-Drosophila H2A.X antibody (Active Motif, #61686) were added per reaction to perform spike-in normalization. The next day, Dynabeads were washed three times with buffer A (140mM NaCl, 1mM EDTA pH 8, 0.5mM EGTA pH 8, 1% Triton X-100, 0.1% SDS, 0.1% sodium deoxycholate, 10mM Tris-HCl pH 8), twice with buffer B (300mM NaCl, 1mM EDTA pH 8, 0.5mM EGTA pH 8, 1% Triton X-100, 0.2% SDS, 0.1% sodium deoxycholate, 10mM Tris-HCl pH 8) and once with buffer C (250 mM LiCl, 10mM Tris-HCl pH 8, 1mM EDTA, 0.5% NP-40, 0.5% sodium deoxycholate). RNA was removed by treatment with RNAse A at 37 °C for 30 minutes and crosslinks were reversed overnight at 65 °C in the presence of Proteinase K and SDS treatment. Eluted DNAs were size selected using SPRIselect beads (Beckman B23319).  RNA-seq: cells were resuspended in 1 ml of Trizol (ThermoFisher, 15596026) and incubated for 5 minutes at room temperature. Then, 0.2 ml of Chloroform (Sigma, C2432) was added and the solution was vortexed vigorously. The mixture was centrifuged at 13,000 rpm for 15 minutes at 4°C, and the upper phase was transferred to a fresh tube. RNA was precipitated using isopropanol (Duksan, 857) and washed with ice-cold 70% ethanol (Sigma, E7023). The precipitated RNA was dissolved in DEPC-treated water (Invitrogen, AM9915G). CUTnTag: The eluted DNA was amplified using 2x PCR master mix (Epicypher, 15-1018) with i5 and barcoded i7 primers. Subsequently, library DNA was purified with SPRIselect beads (Beckman B23319) ).  ChIP-seq: and Libraries were prepared with NEB DNA library prep kit (NEB, E7645L) following the manufacturer's protocol. Libraries were validated using the Tapestation HS D5000 screen tape.  RNA-seq: Library preparation was performed using TruSeq Stranded Total RNA Ribo-Zero H/M/R Gold kit (Illumina, RS-122-2301).