Including a file inside a subdirectory is the same as any other as long as you know the path to the file. Since it’s possible to move the ‘wp-content’ directory, don’t pass that part of the path to the require statement; instead, use the WP_CONTENT_DIR constant.
require_once WP_CONTENT_DIR . '/new-directory/my-file.php';
Also, you can leave out the parentheses since include and require are statements, not functions.
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