The two men, who have pleaded not guilty, will be inside a federal courtroom in Brooklyn on Monday morning.
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Ronald Washington and Karl Jordan Jr. are charged in connection with the shooting that killed Jay back in 2002. Jay was shot inside the hip-hop group's recording studio on Merrick Boulevard in Queens.
Authorities say a third suspect will be tried separately next year.
Over the last two decades, rewards were offered and tips were investigated, but prosecutors say it wasn't until the last five years that the case took crucial strides.
RELATED: 'Set the Record Straight: The Jam Master Jay Case'
Set the Record Straight: The Jam Master Jay Case
They interviewed new people, got witnesses to cooperate, and more ballistic tests were done.
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Prosecutors say the motive for the shooting was likely that Jay was going to cut Washington out of a drug deal.
Jay's family insists he wasn't involved with drugs. The DJ was 37 at the time of his death.
Run DMC made hip-hop more mainstream in the mid-1980s. They became the first rappers with gold and platinum albums, a Rolling Stone cover, and a video on MTV.
If convicted, Washington and Jordan Jr. face at least 20 years in prison.