HEK_SPOC_pSer7_br3
Homo sapiens
Protocols: Cells were harvested, counted, resuspended in 50 mL PBS/108 cells 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, 108 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% Nonidet 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. Triton X-100 was added to a final concentration of 1%. 5-10% of chromatin was kept as an input, to the remainder antibody (Pol II pS5 3E8; Pol II pS2 3E10; Pol II pS7 4E12; total Pol II clone F-12 Santa Cruz sc-55492; TCEA1 Abcam ab185947; H3K27me3 Millipore 07-449) or antiserum (GFP Abcam ab290) was added and rotated ON at 4°C. Protein G or protein A Dynabeads were washed three times in cold block solution (0.5% BSA in PBS), antibody-bound chromatin was added to the beads and rotated 4-6 hours at 4°C. Beads were washed 5 times (8 times for Pol II pS5 ChIP) 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. 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 and NEBNext Multiplex Oligos Primer Set 1-3 (New England Biolabs) according to the manufacturer's instructions.
ENA-FIRST-PUBLIC
2021-02-06T04:08:35Z
ENA-LAST-UPDATE
2020-02-11T10:45:04Z
External Id
SAMEA6534404
INSDC center name
MDC BERLIN
INSDC first public
2021-02-06T04:08:35Z
INSDC last update
2020-02-11T10:45:04Z
INSDC status
public
Submitter Id
E-MTAB-8783:HEK_SPOC_pSer7_br3
broker name
ArrayExpress
cell line
HEK293
common name
human
developmental stage
embryo
disease
normal
genotype
PHF3 SPOC mutant
organism part
kidney
sample name
E-MTAB-8783:HEK_SPOC_pSer7_br3
scientific_name
Homo sapiens