GSM2299469: RNA_Irr_ch_rep_2; Mus musculus; RNA-Seq
Total RNA was isolated with TRIzol reagent (Life Technologies) or Qiagen’s Allprep DNA/RNA/miRNA Universal Kit according to the manufacturer’s protocols Sequencing was performed according to Illumina TruSeq Stranded Total RNA with Ribo-Zero Gold protocol. mRNA was depleted for ribosomal RNA, fragmented and cDNA was synthesized cDNA using SuperScript III Reverse Transcriptase (Thermo Fisher Scientific). cDNA was subjected to a cleanup using AMPure beads (Beckman Coulter) and adenylated to prime for adapter ligation. After a cleanup using AMPure beads DNA fragments were amplified using PCR with the following settings: (30s 98°C; (10s 98°C, 30s 60°C; 30s 72°C) x 9-13 cycles; 5 min 72°C) followed by a final cleanup