ENA first public
2022-02-22
ENA last update
2022-02-22
External Id
SAMEA8920605
INSDC center alias
Tianjin Medical University
INSDC center name
Tianjin Medical University
INSDC first public
2022-02-22T00:26:47Z
INSDC last update
2022-02-22T00:26:47Z
INSDC status
public
Submitter Id
WT E6.5 Epi H3K36me2 rep3
common name
house mouse
geographic location (country and/or sea)
China
isolate
E3.5 or E6.5 Epi
protocol
The star chip-seq assay was performed as Zhang, B. et al. (2016)described . Briefly, embryo samples were lysed in a modified lysis buffer (0.5% NP-40, 0.5% Tween-20, 0.1% SDS and proteinase inhibitor). 0.02 unit of MNase was used for chromatin fragmentation. The reaction was terminated by adding 5 μl Stop buffer (110mM Tris-HCl pH8.0, 55mM EDTA), followed by adding another 45 μl cold “2xRIPA buffer” (1% Triton X-100, 280 mM NaCl, 0.1% SDS, 0.2% DOC, 5 mM EGTA supplemented with proteinase inhibitor). After being spinned at max speed in 4°C for 15 min, the supernatant was transferred to a new tube. Then the sample was supplemented with another 40 μl RIPA buffer (10 mM Tris-HCl pH 8.0, 140 mM NaCl, 1% Triton X-100, 0.1% SDS, 0.1% DOC, 1 mM EDTA). IP sample was then incubated with 1~1.5 μg antibody overnight with rotation at 4°C. On the next day, the sample was incubated with Protein A dynabeads (Life technologies) for 2 hrs with rotation at 4°C. To elute DNA, the beads were resuspended with 27 μl ddH2O and 1 μl 10×Ex-Taq buffer (TaKaRa), 1 μl proteinase K (Roche, 10910000) and incubated at 55°C for 90 min. The supernatant was transferred to a new tube and the proteinase K was inactivated at 72°C for 40 min. The DNA sample was then treated with 1 μl rSAP (NEB, M0371) at 37°C for 1 hr, followed by inactivation at 65°C for 10 min. The resulting sample was subjected to TELP library preparation starting from poly-C tailing as described previously in full detail6 with a slight 5 modification. A modified anchor primer was used for better performance ([BIO]GTTCAGACGTGTGCTCTTCCGATCTGGGGGGGGG, where [BIO] refers to 5'-end biotin). The constructed libraries were sequenced with PE150 on NovaSeq platform.
receipt date
2021-06-18
sample name
WT E6.5 Epi H3K36me2 rep3