ENA first public
2022-02-22
ENA last update
2022-02-22
External Id
SAMEA8547725
INSDC center alias
Tianjin Medical University
INSDC center name
Tianjin Medical University
INSDC first public
2022-02-22T00:26:34Z
INSDC last update
2022-02-22T00:26:34Z
INSDC status
public
Submitter Id
WT-2i H3K36me2 rep3
common name
house mouse
geographic location (country and/or sea)
China
geographic location (region and locality)
TianJin
isolate
mESCs
protocol
ES cells cultured in 2i or SL medium were crosslinked for 10 min with 1% formaldehyde at RT, followed by adding glycine to 0.125M at final concentration and incubation for another 5 min to stop fixation.Then cells were rinsed twice with PBS and cell pellets were resuspended in SDS buffer (1% SDS, 5 mM EDTA, 100 mM NaCl, 0.02% NaN3, 50 mM Tris-HCl, pH 8.1, 1��PIC and 1��PMSF). After the lysis, nuclei were obtained by spinning at 1200 rpm for 6 min and resuspended in appropriate volume of ice-cold IP Buffer (SDS Buffer: Triton Dilution Buffer (100 mM Tris-HCl, pH 8.6, 100 mM NaCl, 5 mM EDTA, 5% Triton X-100, 0.02% NaN3, 1��PIC and 1��PMSF) =1:1) for sonication. The sonication was optimized to enrich chromatin fragments with an average length of 200-500 bp. If an optimal sonication was achieved, the chromatin samples were diluted with IP buffer to a desired concentration. Then primary antibodies were added and incubated overnight at 4��C rotating. On the next day, 30-40 ��l of protein A/G magnetic beads were add and incubate 2-4 hrs at 4��C. Then the beads were washed one with 150 mM wash buffer (1% Triton X-100, 0.1% SDS, 150 mM NaCl, 2 mM EDTA, pH 8.0, 20 mM Tris-HCl, pH 8.0) and three times with 500 mM wash buffer (1% Triton X-100, 0.1% SDS, 500 mM NaCl, 2 mM EDTA, pH 8.0, 20 mM Tris-HCl, pH 8.0). After the washes, the beads were added 120 ��l of de-crosslink buffer and incubated 3 hrs up to overnight at 65��C (shaking at 1,100 rpm) to elute DNA from beads. Then the input and ChIP DNA was purified with QIAGEN PCR purification kit.
receipt date
2021-04-09
sample name
WT-2i H3K36me2 rep3