Total RNA was purified with Trizol (Thermo Fisher Scientific) following the supplier’s recommended method and Poly-A selected using the MicroPoly(A) Purist Kit (Ambion). Samples were processed according to the manufacturer’s protocol. Subsequently, libraries were prepared following the dUTP protocol37. Sequencing reads (100 bp) were generated on Hi-Seq 2000 Illumina platforms. Given that the samples contained a mixed population of human and mouse cells, paired-end reads were aligned to both the human reference sequence NCBI Build hg19 and the mouse reference sequence NCBI Build 37/mm9 with TopHat (v1.1.3)38. Properly paired reads were extracted using the samtools function. Only properly paired reads to the human reference were considered for subsequent analysis. Expression levels of RefSeq-annotated genes were calculated in units of reads per kilobase of exon model per million mapped fragments (RPKM). Genes with low RPKM (average log2 RPKM across all samples less than 1) were removed. Differential expression analysis was performed using Students’ t-test function “t.test” in R, and genes with P value < 0.05 and at least two-fold expression change were defined as significant.
