title: HEK_KO_input_br2
organism: Homo sapiens
description: Protocols: Cells were harvested and counted, 108 cells were resuspended in 50 mL PBS and fixed for 10 minutes by adding formaldehyde to a final concentration of 1%. Formaldehyde was quenched by adding glycine pH 3 to a final concentration of 0.6 M for 15 minutes. Cells were centrifuged and washed twice in cold PBS. To isolate nuclei, fixed cells were resuspended in 5 mL cold lysis buffer 1 (50 mM HEPES/KOH pH 7.5, 140 mM NaCl, 1 mM EDTA, 10% glycerol, 0.5% Nonident P-40, 0.25% Triton X-100, 1x protease inhibitor; for Pol II ChIP 2 mM Na3VO4 and 2 mM NaF), rotated for 10 min at 4°C and centrifuged. Nuclei were resuspended in 5 mL cold lysis buffer 2 (10 mM Tris-Cl pH 8, 200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 1x protease inhibitors; for Pol II ChIP 2 mM Na3VO4 and 2 mM NaF), rotated for 10 min at room temperature and centrifuged. The pellet was resuspended in 3 mL lysis buffer 3 (10 mM Tris-Cl pH 8, 100 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 0.1% Na-deoxycholate, 0.5% N-lauroylsarcosine, 1x protease inhibitors; for Pol II ChIP 2 mM Na3VO4 and 2 mM NaF).   Chromatin was sheared to an average size of 200-600 bp using the Bioruptor Pico (Diagenode) for 20 cycles 30 sec on/30 sec off. Sheared chromatin was centrifuged at maximum speed for 10 min at 4°C to remove insoluble debris. Triton X-100 was added to a final concentration of 1%. 5-10% of chromatin was kept as an input, the remainder was split into 3 parts. To each aliquot 10 µg of antibody (Pol II antibodies: CTD pS5 4H8 Abcam ab5408, CTD pS2 Abcam ab5095) or 2 µL of antiserum (GFP Abcam ab290) were added and rotated ON at 4°C. 75 µL protein G or protein A Dynabeads per ChIP reaction were washed three times in cold block solution (0.5% BSA in PBS; Pol II ChIP) or PBS (PHF3-GFP ChIP), antibody-bound chromatin was added to the beads and rotated 4-6 hours at 4°C. Beads were washed 5 times in RIPA washing buffer (50 mM HEPES/KOH pH 7.5, 500 mM LiCl, 1 mM EDTA, 1% NP-40, 0.7% Na-deoxycholate) and once in 50 mM NaCl in TE. Crosslinked protein-DNA complexes were eluted in 200 µL elution buffer (50 mM Tris-Cl pH 8, 10 mM EDTA, 1% SDS) for 15 min at 65°C. Crosslinks were reversed at 65°C ON. RNA was degraded by adding 0.2 mg/mL RNase A for 2 h at 37°C, proteins were digested by adding 0.2 mg/mL proteinase K and 5.25 mM CaCl2 for 30 min at 55°C.   . For immunoprecipitation of exogenously expressed FLAG-PHF3 constructs, a 10 cm dish of transfected HEK293T cells was used. Cells were harvested 48 h after transfection and lysed in lysis buffer (50 mM Tris-Cl pH 8, 150 mM NaCl, 1% Triton, 1x protease inhibitors, 2 mM Na3VO4, 1 mM PMSF, 2 mM NaF, 50 units/mL benzonase and 1 mM DTT) for 1 h at 4°C. 10% of the cleared lysate was kept as input and the rest was incubated for 2 h on a rotating wheel at 4°C with anti-FLAG M2 magnetic beads (Sigma). For Pol II IP, Protein G Dynabeads (Invitrogen) were washed twice with TBS and incubated with 5 μg of mouse anti-FLAG M2 (Sigma), rabbit anti-pS2 Pol II (Abcam ab5095), mouse anti-pS5 Pol II 4H8 (Abcam ab5408) or rat anti-pS7 Pol II clone 4E12 (Millipore) antibodies for 1 h on a rotating wheel at room temperature. Beads were washed twice with TBS and cleared lysates were added for immunoprecipitation on a rotating wheel at 4°C ON. For immunoprecipitation of endogenously tagged PHF3-GFP, two 15 cm dishes were used for each cell line. Cells were harvested and lysed in lysis buffer (as above but without DTT). The lysates were incubated on a rotating wheel at 4°C ON with rabbit anti-GFP (Abcam ab290) antibody. The samples were added to protein G Dynabeads (Invitrogen) and incubated on a rotating wheel at 4°C for 6 h. Beads were subsequently washed three times with TBS and immunoprecipitated proteins were eluted twice with 0.1 M glycine pH 2 and neutralized with Tris-Cl pH 9.2. For Western blot, 2% of the input and 20% of the eluate were loaded for each sample. For mass spectrometry analysis of PHF3 interactome and PTMs, anti-FLAG immpunoprecipitations were performed with anti-FLAG M2 magnetic beads as above and samples were processed for on beads or gel digestion as previously described DNA was purified by phenol-chloroform extraction, ethanol-precipitated and resuspended in 50 µL nuclease-free water.  Next generation sequencing libraries were prepared using the NEBNext Ultra II DNA library Prep Kit for  Illumina (New England Biolabs) according to the manufacturer's instructions.
attributes.tag: isolate
attributes.value: not applicable
attributes.tag: organism
attributes.value: Homo sapiens
attributes.tag: developmental stage
attributes.value: embryo
attributes.tag: disease
attributes.value: normal
attributes.tag: organism part
attributes.value: kidney
attributes.tag: cell_type
attributes.value: epithelial cell
attributes.tag: cell_line
attributes.value: HEK293
attributes.tag: genotype
attributes.value: CRISPR/Cas9 mediated PHF3 knockout
attributes.tag: ENA-FIRST-PUBLIC
attributes.value: 2019-12-02
attributes.tag: ENA-LAST-UPDATE
attributes.value: 2018-12-10