description: ES cells cultured in 2i or SL medium were used for ChIP-Seq
attributes.tag: ENA first public
attributes.value: 2022-02-22
attributes.tag: ENA last update
attributes.value: 2022-02-22
attributes.tag: External Id
attributes.value: SAMEA8547764
attributes.tag: INSDC center alias
attributes.value: Tianjin Medical University
attributes.tag: INSDC center name
attributes.value: Tianjin Medical University
attributes.tag: INSDC first public
attributes.value: 2022-02-22T00:26:35Z
attributes.tag: INSDC last update
attributes.value: 2022-02-22T00:26:35Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: WT-SL Brg1 rep1
attributes.tag: common name
attributes.value: house mouse
attributes.tag: geographic location (country and/or sea)
attributes.value: China
attributes.tag: geographic location (region and locality)
attributes.value: TianJin
attributes.tag: isolate
attributes.value: mESCs
attributes.tag: protocol
attributes.value: 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.
attributes.tag: receipt date
attributes.value: 2021-04-09
attributes.tag: sample name
attributes.value: WT-SL Brg1 rep1