RNA-Seq of human breast metastatic tumor: Sample 016-009M-METASTATIC
For RNA purification, three 10-μm FFPE slides were cut for each tumor, and macrodissection was performed, when needed, to avoid normal breast contamination. RNA was isolated from primary and metastatic paired FFPE tumor using QUIAGEN RNeasy Mini Kit (Cat No. 74104). RNA was quantified using a NanoDrop® Spectrophotometer. The quality of RNA was  then  analyzed  via  2100 Bioanalyzer Instrument (Agilent)  using the  RNA 6000 Nano  assay (Agilent)  for determination  of  an  RNA  Integrity  Number  (RIN). Gene expression profiles from primary and metastatic tumors were generated by mRNA-sequencing using an Illumina HiSeq 2000 (Agilent). Briefly, 1 μg of total RNA was converted to RNAseq libraries using the TruSeq Stranded Total RNA Library Prep Kit with Ribo-Zero Gold (Illumina) and sequenced on an Illumina HiSeq 2000 using a 2x50bp configuration with an average of 136 million read pairs per sample. Quality-control-passed reads were aligned to the human reference CGRh38/hg38 genome using Spliced Transcripts Alignment to a Reference (STAR) software (https://github.com/alexdobin/STAR). The alignment profile was determined by Picard Tools v1.64.