GSM3912503: WT Mettl3 IP r2; Mus musculus; ChIP-Seq
Sample information curated by ChIP-Atlas
Antigen
Antigen Class
TFs and others
Antigen
Mettl3
Cell type
Cell type Class
Pluripotent stem cell
Cell type
ES cells
NA
NA
Attributes by original data submitter
Sample
source_name
Embryonic stem cells
cell type
mouse embryonic stem cells
strain
C57BL/6
antibody
Rabbit anti-METTL3 (ab195352, Abcam)
Sequenced DNA Library
library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
The cells were then collected in lysis buffer (0.1% SDS, 2 mM EDTA, 1 x protease inhibitors, 20 mM Tris–HCl, pH 8.0, 150 mM NaCl, 1% Triton) and the lysates were sonicated by a Bioruptor UCD-200 (Diagenode) to result in DNA fragments of 200 to 500 bp in length. Cellular debris was removed by centrifugation and the lysates were diluted 1:10 in ChIP dilution buffer (0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA, 167 mM NaCl, 1 x protease inhibitors, 16.7 mM Tris–HCl, pH 8.0). Take 1% of sonicated chromatin as Input control. Washed Protein G Magnetic beads (Invitrogen) two times with ChIP buffer, eluted beads in 1 mL of ChIP buffer, added METTL3 antibody (Abcam) and incubated the mixture 2 hours on rotating platform at 4 °C. The washed the antibody-coated beads with ChIP buffer three times. Chromatin solutions were incubated overnight at 4 °C with rotation with antibodies-coated beads. The beads were washed sequentially for 3 min by rotation with 1 ml of the following buffers: low salt wash buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 150 mM NaCl, 20 mM Tris–HCl, pH 8.0), high salt wash buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 500 mM NaCl, 20 mM Tris–HCl, pH 8.0) and LiCl wash buffer (0.25 M LiCl, 1% Nonidet P-40, 1% sodium deoxycholate, 1 mM EDTA, 10 mM Tris–HCl, pH 8.0). Finally, the beads were washed twice with 1 ml TE buffer (1 mM EDTA, 10 mM Tris–HCl, pH 8.0). The immuno-complexes and Input were then eluted by adding 400 μl of elution buffer (100 mM NaHCO3, 1% SDS) plus 18 μl 5 M NaCl and incubating for 4 hrs at 65 °C to reverse protein-DNA crosslinks. The remaining proteins were digested by adding 5 μl of proteinase K (Promega) and incubating overnight at 65 °C. The DNA was recovered by phenol/chloroform/isoamyl alcohol (25:24:1) extractions and precipitated with 0.1 volume of 3 M sodium acetate, pH 5.2, and 3 volumes of ethanol using glycogen as carrier. Library preparation was performed by using KAPA HyperPlus Kits (Kapabiosystems) according to the manufacturer's protocols.