Table 2.

Comparison of the linker sequences in iMARGI¹⁵, GRID-seq¹⁷, and ChAR-seq¹⁶ ssDNA: single strand DNA. dsDNA: double strand DNA.

	iMARGI	GRID-seq	ChAR-seq	Comment
Biotinylation	Biotinylated	Biotinylated	Biotinylated	Allows for selection of ligation products.
Strands	A longer ssDNA (top strand) annealed with a shorter ssDNA (top strand)	ssDNA annealed with an RNA-DNA chimeric sequence	dsDNA with a 5’-overhang and a 3’-three carbon spacer	GRID-seq’s linker sequence requires special synthesis
5’ adenylation	5’ adenylated	5’ adenylated	5’ adenylated	For ligation to 3’-end of RNA
5’ random bases	Contains 2 random bases on the 5’ of linker top strand	None	None	5’ random bases (NN) in the linker top strand can minimize biases of RNA ligase.
Restriction sites	BamHI	2 × Mmel	Dpn II	Dpn II allows for carrying out Hi-C experiments in parallel.
Complementarity to adaptors	Yes	No	No	Without sequencing the linker sequence, iMARGI is able to resolve the orientation of the RNA-linker-DNA. There is no need to ligate library construction adaptors in iMARGI.