Single Cell Hi-C Library Construction via Sticky End Ligation
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Solution Overview
Problem
Current Hi-C methods are not suitable for small sample sizes, including single cells, due to the requirement for large initial sample sizes and inefficient processes that result in significant loss of chromatin interaction information, leading to low resolution and inability to detect individual cell chromatin conformation differences.
Innovation Solution
A method for constructing a Hi-C library that starts with a small amount of cells (1-10,000 cells) using sticky end restriction enzymes for digestion, eliminating biotin labeling and blunt end ligation, and employing random fragmentation for higher resolution, allowing for the construction of a sequencing library from single cells or trace samples.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Loss of information
If traditional Hi-C methods use formaldehyde cross-linking and blunt end ligation with biotin labeling, then chromatin structure can be captured, but the process requires large sample sizes (≥10^6 cells) and results in significant loss of chromatin interaction information
Solution Approach 1:
The invention extracts and removes the biotin labeling step and blunt end ligation step from the traditional Hi-C protocol. By eliminating these steps, the method reduces chromatin interaction information loss while enabling application to small sample sizes (1-10,000 cells), directly resolving the contradiction between information preservation and sample size reduction
Solution Approach 2:
The invention changes the ligation method from blunt end ligation to sticky end ligation, and modifies the restriction enzyme digestion parameters to use sticky end enzymes. This parameter change improves ligation efficiency and reduces information loss, enabling the method to work effectively with small sample sizes while preserving chromatin interaction information
2Measurement precision
If traditional Hi-C methods use blunt end ligation, then ligation can be performed, but the resolution is low and chromatin interaction information is lost
Solution Approach 1:
The invention changes the ligation type from blunt end to sticky end ligation, and uses sticky end restriction enzymes for digestion. This parameter change increases ligation efficiency and resolution, enabling precise detection of chromatin conformations while preserving interaction information, thus resolving the contradiction between measurement precision and information loss
3Ease of operation
If Hi-C analysis is performed on single cells or trace samples, then individual cell chromatin conformation can be detected, but traditional methods cannot process such small samples
Solution Approach 1:
The invention removes the biotin labeling and blunt end ligation steps that are incompatible with single-cell analysis. This extraction enables the method to process single cells and trace samples while maintaining detection accuracy through improved sticky end ligation efficiency, resolving the contradiction between ease of operation on single cells and detection reliability
Solution Approach 2:
The invention optimizes the ligation conditions and uses sticky end enzymes with parameters suited for low-input samples. This enables reliable detection of chromatin conformations in single cells by improving reaction efficiency at low sample concentrations, resolving the contradiction between single-cell applicability and detection accuracy
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
This approach enables high-resolution, high-information-content Hi-C analysis on small samples, including single cells, with improved efficiency and reduced information loss, facilitating the detection of chromatin conformation differences between cells.
Implementation Method 1
digest the fixed chromatin with a restriction enzyme MboI to obtain fixed chromatin fragments
Implementation Method 2
the sticky end fragments are reconnected directly to obtain the reconstructed fixed chromatin fragments
Implementation Method 3
the cross-linking is reversed by high temperature treatment (65°C) to release double-stranded DNA molecules
Implementation Method 4
the DNA is subjected to a polymerase chain reaction to obtain amplification products
Data Source
AI summary
Provided in the present invention is a method for constructing a high-resolution single cell Hi-C library with a large amount of information, comprising the following steps: Step B: obtain a small amount of fixed chromatin; Step C: digest the fixed chromatin in Step B to obtain fragments of the fixed chromatin; Step D: reconnect the fragments of the fixed chromatin in Step C directly to obtain reconnected fragments of the fixed chromatin; Step E: de-fix the reconnected frag ments of the fixed chromatin in Step D to release DNA fragments; Step F: amplify the released DNA fragments in Step E to obtain amplified products; and Step H: construct a sequencing DNA library by using the amplified products as the DNA fragments to be sequenced.