Dec 04, 2024

Public workspaceChIP-seq protocol

DOI
dx.doi.org/10.17504/protocols.io.rm7vzkb62vx1/v1
  • 1Sloan Kettering Institute;
  • 2Johns Hopkins University
  • IGVF
Michael Beer
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DOI: dx.doi.org/10.17504/protocols.io.rm7vzkb62vx1/v1
Protocol Citation: Renhe Luo, Michael Beer, Danwei Huangfu 2024. ChIP-seq protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.rm7vzkb62vx1/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: December 03, 2024
Last Modified: December 04, 2024
Protocol Integer ID: 113702
Funders Acknowledgements:
NHGRI
Grant ID: HG012051
Abstract
ChIP-seq protocol for ESC-DE differentiation
ChIP-seq protocol
ChIP-seq protocol
For each sample, around 30 million cells were crosslinked with 1% formaldehyde and quenched with 0.125M glycine.
Fixed cells were then lysed in 700uL SDS buffer (1% SDS, 10 mM EDTA, 50 mM Tris–HCl, pH 8), and incubated for 10min on ice.
Sonication was performed on a Branson Sonifier 150 set at 30% amplitude for 5min30s total on (10s on/off pulsing).
Clear supernatant was collected for antibody binding overnight, followed by Dynabeads (Thermo Fisher Scientific;10004D) incubation for 6 hours at 4°C.
Then the beads were pelleted and washed twice with low salt (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris–HCl, pH 8, 150 mM NaCl), high salt (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris–HCl, pH 8, 500 mM NaCl), and TE buffer (10 mM Tris–HCl, pH 8, 1 mM EDTA) respectively.
The DNA was eluted from the beads by incubating in elution buffer (1% SDS, 0.1 M NaHCO3) at 65°C for 15min and decrosslinked with 5M NaCl at 65°C overnight.
A total of 10 μl 0.5 M EDTA, 20 μl 1 M Tris–HCl, pH 6.5, and 1 μl Proteinase K (20 mg ml−1) were added to decrosslinked product and incubated for 1 h at 45°C.
DNA was isolated by using QIAquick PCR purification kit (Qiagen, 28104; QIAGEN).
Then the sequencing library was generated by using the NEBNext® Ultra II DNA Library Prep Kit (New England Biolabs; E7103S) and NEBNext® Multiplex Oligos for Illumina® (New England Biolabs Index Primers Set 1; NEB, E7335S).
Samples were pooled and submitted to MSKCC Integrated Genomics Operation core for quality control and sequencing on Illumina HiSeq 4000 platform.