Whole transcriptome profiling of murine Hic-5 HET and KO Cancer Associated Fibroblasts (CAFs)

To investigate the role of the differential expression of Hydrogen peroxide-inducible clone 5 (Hic-5) gene on CAFs, primary tumor cells from MMTV-PyMT mice that were heterozygous (HET) or knockout (KO) for the Hic-5 gene were freshly isolated and grown in culture. RNA was isolated using Trizol reagent. Sequencing libraries were prepared with the TruSeq Stranded Total RNA Library Prep Kit RiboZero Gold (Illumina), using 1 microgram of total RNA as input. Libraries were pooled and sequenced on the NextSeq 500 instrument (Illumina), with a single end 1 x 75 bp reads using a High Output cycle reagent kit. FASTQ files were processed to remove adapter sequences and aligned using Bowtie2 version 2.2.5 to the mouse mm10 genome and quantified to Ensembl Transcripts release 86. Data were normalized by RPKM. Differential gene expression analyses were performed in Partek Flow using Gene Specific Analysis (GSA) method. Overall design: Differential expression of technical triplicates of cultured mouse CAFs with either Hic-5 heterozygous (HET) or Hic-5 knockout (KO) genotypes.