Hi Ying, One thing I am concerned is that my files

is kind of large, e.g., for each end I got a file with 20.7 gb, so totally 41.4 gb for both ends of this sample. So do you think it is because the files are too big?

You'll want to use the FTP upload process to upload data this large to Galaxy. See the upload page for details; we recommend FileZilla as a nice GUI for performing FTP: http://filezilla-project.org/

Also when the genome center sent me the data, they already separate the two ends into two files, so I uploaded two files for one sample, but when do tophat analysis, do I need to merge those two files into one file?

You'll want to perform a single Tophat analysis on both files since they're paired. However, no merging is required: Tophat will accept both files in a single run; see the Tophat tool for details.

do you know what is the usual parameter set up for paried-end RNA-seq analysis(with a 300 bp fragment)?

Tophat has a set of default parameters, but you'll likely want to experiment with different values to see what works best for your data. Note that the mean inner distance between pairs is (Insert_size-N*2 ) where N is the size of each read fragment. Best, J.