GSM8820627: primary cortical neurons, 24hrs post 4 rounds of 1hr ETP treatment, rep2, Npas4; Mus musculus; OTHER
Approximately 10 million primary cortical neurons (DIV13) or Hek 293T cells were cross-linked in 2% formaldehyde for 10 min at room temperature and quenched with glycine (0.13M final concentration). Cells were incubated for 20 min on ice in lysis buffer (50mM Tris-HCl pH 75, 150mM NaCl, 5mM EDTA, 0.5% NP-40, 1% TX-100, and protease inhibitors). Nuclei were resuspended in 450uL 1.2x CutSmart buffer and permeabilized with 15uL 10% SDS for 1hr and 75uL 20% Triton X-100. A sample of undigested chromatin was collected to assess chromatin integrity. DNA was digested with 100U NlaIII at 37°C for 3hrs while shaking at 750rpm and then an additional 100U of NlaIII was added and incubated overnight. The extent of chromatin digestion was assessed via gel electrophoresis. NlaIII was deactivated for 20 min at 65°C. Chromatin was diluted to 7mL, supplemented with 10X ligation buffer, and 4000U NEB T4 DNA Ligase for overnight ligation at 16°C while shaking. Ligation efficiency was checked by gel electrophoresis. Crosslinks were reversed and proteins digested with proteinase K (30uL of 10mg/ml) overnight at 65°C. DNA was extracted via phenol:chloroform and precipitated with ethanol. DNA was then digested overnight at 37°C with 50U DpnII supplemented with 10X DpnII Buffer while shaking. Second digestion efficiency was determined via gel electrophoresis. DpnII was deactivated at 65°C for 20 min. DNA circularized in a 7mL reaction, supplemented with 10X ligation buffer, and 4000U NEB T4 DNA Ligase overnight at 16°C with shaking. Indexed Illumina sequencing adapters were introduced to ligation fragments of interest with a two-step PCR strategy using Expand Long Template Polymerase starting with 200ng of 4C library template. Viewpoint-specific primers were used to amplify circular templates with the bait of interest. The first round of amplification with the following thermocycler conditions 94°C  for 2 min; 94°C for 10sec, 58° C for 1 min, 68°C for 3 min, for 16 cycles; and 68°C for 5 min. Five different PCR reactions per sample were pooled and 50uL of the pool was used to perform 0.8 x AMPure XP bead purification. 5uL of purified round 1 PCR product was then used for the second round of amplification conducted with the following cycling conditions 94°C for 2 min; 94°C for 10 sec, 60°C for 1 min, 68°C for 3 min for 20 cycles; and 68°C for 5 min. PCR clean up was doing using Qiagen PCR Purification.  DNA fragment size of final, clean PCR products was assessed via Tapestation (Agilent). Library concentrations for each sample was assessed using fluormetry (dsDNA high sensitivity Qubit assay, Thermo) and RT-qPCR (KAPA Biosystems) library quantification.