Spin column based rna purification kit

Total RNA was extracted and DNAse-I treated using a spin column-based RNA purification kit (Macherey-Nagel). cDNA was prepared with SuperScript II reverse transcriptase (Invitrogen). Primers (listed in Supplemental Experimental Procedures) were used for SYBR Green qPCR (Applied Biosystems), and specificity was confirmed with dissociation curves. For mRNA sequencing, 100-bp single-end RNA-seq libraries were prepared using the Illumina TruSeq Stranded mRNA reagents (Illumina). Cluster generation was performed with the resulting libraries using the Illumina TruSeq SR Cluster Kit v4 reagents. Sequencing was performed with Illumina HiSeq 2500 in 100-bp reads run. The RNA-seq reads were mapped to the mm9 genome using TopHat (Kim et al., 2013 (link)), allowing multimapped reads to be randomly assigned once among the mapped loci. Gene counts were generated with HTseq-count program using default parameters, and TE counts were computed using BEDtools (multicov). Sequencing depth normalization and differential expression analyses were performed using the voom function of Bioconductor package LIMMA (Law et al., 2014 (link)).