RNA-Seq of Mus-musculus BAT: male KIKO 21 C
Total RNAs have been checked on Caliper GX (PerkinElmer, Waltham, MA) to evaluate the integrity. 1.3 ug of good quality RNAs (R.I.N. > 7) have been used as input of the TruSeq Stranded mRNA Sample Prep kit (Illumina, San Diego, CA) for library preparation following the manufacturers instructions. The poly-A mRNA was fragmented 3 minutes at 94C and the purification step after pcr-enrichment has been performed by using 0.8X Agencourt AMPure XP beads.Final libraries were quantified by using the Qubit 2.0 Fluorometer (Invitrogen, Carlsbad, CA) and quality tested by Agilent 2100 Bioanalyzer High Sensitivity or DNA 1000 assay (Agilent Technologies, Santa Clara, CA). Libraries were then processed with Illumina cBot for cluster generation on the flowcell, following the manufacturers instructions and sequenced on paired-end mode on HiSeq2500 (Illumina, San Diego, CA). The CASAVA 1.8.2 version of the Illumina pipeline was used to processed raw data for both format conversion and de-multiplexing.Both starting RNA samples and final libraries were quantified by using the Qubit 2.0 Fluorometer (Invitrogen, Carlsbad, CA) and quality tested by Agilent 2100 Bioanalyzer RNA Nano assay (Agilent technologies, Santa Clara, CA). Libraries were then processed with Illumina cBot for cluster generation on the flowcell, following the manufacturers instructions and sequenced in multiplexing producing the desired million reads per sample on HiSeq2500 (Illumina, San Diego, CA). The CASAVA 1.8.2 version of the Illumina pipeline was used to processed raw data for both format conversion and de-multiplexing.