title: Disruption of ADNP-KDM1A-GTF2I complex drives neural differentiation imbalance in Helsmoortel-Van der Aa syndrome [ ChIP-seq ]
design: Disruption of ADNP-KDM1A-GTF2I complex drives neural differentiation imbalance in Helsmoortel-Van der Aa syndrome [ ChIP-seq ]
library_construction_protocol: Approximately 10e-7 cells were released with Accutase and then crosslinked with formaldehyde 1% in PBS 1X (Sigma, F8775) for 8 minutes at room temperature (RT) in rotation. Crosslinking was stopped by adding glycine at a final concentration of 0.125 mM and incubation on ice for 5 minutes. Cells were pelleted by centrifugation at 500g for 3 min at 4°C and the pellet was washed once in ice cold PBS.  Cells were lysed in 10 mL buffer A (50 mM HEPES pH 8.0, 140 mM NaCl, 1 mM EDTA, 10% glycerol, 0.5% NP-40, 0.25% Triton X-100) for 10 minutes on ice. After centrifugation the pellet was resuspended in 5 mL buffer B (10 mM Tris pH 8, 1 mM EDTA, 0.5 mM EGTA and 200 mM NaCl) and incubated for 5 min on ice. Then nuclei were pelleted by centrifugation at 500 g for 3 min at 4°C, resuspended in 150 µL buffer C (50 mM Tris pH 8, 5 mM EDTA, 1% SDS, 100 mM NaCl, 1x Roche complete mini protease inhibitors) and incubated on ice for 10 min. Then 3.5 mL ice cold TE buffer was added and chromatin was sheared in 13 mL tubes in a Branson Sonifier 450 (Marshall Scientific) device for 4 pulses (30 s ON / 30 s OFF) at 30% amplitude. Then 350 μL 10x ChIP buffer (0.1% SDS, 10% Triton X-100, 12 mM EDTA, 167 mM Tris-HCl pH 8, 1.67M NaCl) was added, chromatin was transferred into 2 mL Eppendorf tubes and spun for 10 min at 13000 g, 4°C. 1% sheared chromatin was used as control (input) for peak calling and downstream analyses, the rest was transferred into fresh tubes.5 µg of antibody were used for H3K27ac (Abcam, ab4729), H3K4me1 (Abcam, ab8895), and H3K9me3 (Abcam, ab8898) IPs while 10 µg were used for ADNP (Sigma-Aldrich, F1804 monoclonal anti-FLAG M2) and CTCF (Cell Signaling, #2899) IPs. Samples were incubated O/N on a rotating wheel at 4°C. The next morning, 40 µL Protein G Dynabeads (mixed 1:1 and washed with 1x ChIP buffer) were added and incubation was continued for another 4 h. ChIPs were washed for 1 minute each with 4x LSB (10 mM Tris-HCl pH 8.0, 1 mM EDTA pH 8.0, 140 mM NaCl, 1% Triton X-100, 0.1% SDS, 0.1% Na-deoxycholate), 1x HSB (10 mM Tris-HCl pH 8.0, 1 mM EDTA pH 8.0, 360 mM NaCl, 1% Triton X-100, 0.1% SDS, 0.1% Na-deoxycholate), 2x LiSB (10 mM Tris-HCl pH 8.0, 1 mM EDTA, pH 8.0, 250 mM LiCl, 0.5% NP-40, 0.5% Na-deoxycholate), 1x TEplus (10 mM Tris-HCl pH 8.0, 1 mM EDTA, 50 mM NaCl). Beads were transferred to a fresh tube during the last wash and wash buffer was completely removed before adding 100 µL elution buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA pH 8.0, 150 mM NaCl, 1% SDS) and incubating 30 minutes at 65°C with constant shaking. Elution was repeated once more with 50 µL elution buffer for 10 minutes and eluates were pooled, 2 μL RNaseA (20 µg/µL) were added and samples were incubated for 1 h at 37°C. Input samples were adjusted to 150 µL total volume with elution buffer and processed similar to ChIP samples. Then 2 µL Proteinase K (20mg/ml) was added and samples were incubated 2 h at 55°C and then O/N  at 65°C. 2X volumes of AMPure XP beads and 1 volume Isopropanol was added and samples were vigorously mixed and incubated for 10 min at RT. Then beads were washed twice with 80% EtOH and DNA was eluted in 30 µL 10 mM Tris pH8.0 for 5 min at 37°C. DNA libraries were prepared by Genomic Unit at the IFOM/IEO/IIT campus according to manufacturer protocols, and sequenced on the Illumina Novaseq 6000 instrument at 50 bp paired-end read length and a coverage of 35 million reads per sample. 