All RNA samples were subjected to RNA sequencing (RNAseq; HudsonAlpha Genomic Services Lab, Huntsville, AL) as performed before [37 (link)]. In short, total RNA concentration was estimated by Qubit 2.0 Fluorometer (Invitrogen, Carlsbad, CA, USA) and RNA integrity by using the Agilent 2100 Bioanalyzer (Applied Biosystems, Carlsbad, CA, USA). RNAseq libraries were formed from approximately 500 ng total RNA of each pooled sample, followed by poly(A) enrichment. RNAseq was performed using paired-end sequencing on Illumina HiSeqH2000 (Illumina, San Diego, CA, USA), at 50 base pairs, generating over 25 million paired reads per sample. Raw RNAseq FASTQ files were demultiplexed by bcl2fastq conversion software v1.8.3 (Illumina, Inc., San Diego, CA, USA) using default settings.
RNAseq data was analyzed using GeneSifter software (VizX Labs, Seattle, WA). RNAseq reads were mapped to the Mus musculus reference genome build 37.2, and for this, the reads were trimmed by 15 base pairs at the five-prime end. Subsequently, transcript abundance was calculated by estimating the reads per kilobase of exon per million mapped reads (RPKM), and normalization to the number of mapped reads was used for comparison of two mRNA sets. A t test was used for pairwise comparison and a likelihood ratio test to adjust for distribution probability.
RNAseq data was analyzed using GeneSifter software (VizX Labs, Seattle, WA). RNAseq reads were mapped to the Mus musculus reference genome build 37.2, and for this, the reads were trimmed by 15 base pairs at the five-prime end. Subsequently, transcript abundance was calculated by estimating the reads per kilobase of exon per million mapped reads (RPKM), and normalization to the number of mapped reads was used for comparison of two mRNA sets. A t test was used for pairwise comparison and a likelihood ratio test to adjust for distribution probability.
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