MR600D Standardized ChIP Chromatin Shearing Workflow

MR600D ChIP Chromatin Shearing Workflow

1. Purpose and Principle

Chromatin shearing is a critical step for ChIP, ChIP-qPCR, and ChIP-seq. This workflow uses the MR600D non-contact ultrasonic disruptor to fragment cross-linked chromatin gently and uniformly while preserving protein-DNA interactions. The target fragment range is generally 200-1000 bp, with 200-500 bp preferred for ChIP-seq and 500-1000 bp acceptable for many ChIP-qPCR validation workflows.

2. Sample and Reagent Requirements

• Sample types: adherent cells, suspension cells, and soft animal tissues such as liver, kidney, or brain tissue.
• Cross-linking: 1% formaldehyde for about 10 min at room temperature, followed by 0.125 M glycine quenching for 5 min.
• Cell input: typically 1 x 10^6 to 5 x 10^6 cells per sample.
• Reagents: SDS lysis buffer, dilution buffer, PMSF, protease inhibitors, phosphatase inhibitors, nuclease-free water, and nuclease-free tubes.
• Device condition: MR600D pre-cooled to 4 C with stable water circulation between 3 C and 6 C.

3. Standard Workflow

1. Wash cells twice with PBS after cross-linking and quenching.
2. Resuspend nuclei in 100-500 ul SDS lysis buffer with fresh inhibitors.
3. Lyse on ice for about 10 min and remove bubbles completely.
4. Place tubes symmetrically in the MR600D rack, keeping the energy field balanced.
5. Run intermittent sonication under low-temperature circulation.
6. Centrifuge at 12000 rpm, 4 C for 10 min and collect the supernatant as sheared chromatin.

4. Reference Shearing Conditions

Workflow	Pulse mode	Total time	Target range	Notes
ChIP-seq grade	15 s on / 15 s off	300-600 s	200-500 bp	Common for high-resolution sequencing
ChIP-qPCR validation	15 s on / 15 s off	300-600 s	500-1000 bp	Suitable for many enrichment checks
Tissue samples	15 s on / 15 s off	Increase 30-50%	Depends on tissue	Keep temperature unchanged

5. QC and Optimization

QC can be performed after reverse cross-linking using agarose gel electrophoresis or a fragment analyzer. Avoid bubbles, over-cross-linking, insufficient lysis, and unstable water temperature. If fragments are too large, extend total time or improve lysis. If fragments are too small or IP signal is weak, reduce processing time, lower energy exposure, or check sample quality and antibody compatibility.

6. Summary

MR600D helps ChIP workflows by delivering uniform, low-temperature, non-contact chromatin shearing while reducing local overheating and probe contamination. It is suitable for ChIP-qPCR method setup and ChIP-seq library preparation.