GSM1787276: mouse ZT8_control_12; Mus musculus; RNA-Seq
Liver samples were quickly excised every four hour for 20 hours and snap-frozen in liquid nitrogen. Mouse liver (50 - 100 mg) was homogenized using magnetic beads and a TissueLyzer. Total RNA was isolated using Qiagen RNeasy Kit and the RNA integrity was checked using an Agilent Technologies 2100 Bioanalyzer. Illumina TruSeq RNA Sample Prep v2 was used for library construction, according to manufacturer's instructions. Briefly, poly-A containing mRNA was isolated using poly-T oligo-attached magnetic beads and fragmented during elution. First strand cDNA was reverse transcribed from cleaved RNA fragments primed with random hexamers. Second strand cDNA was synthesized and isolated using Ampure XP beads. Blunt ends were created using 3' to 5' exonuclease activity and polymerase activity. A single "A" nucleotide was added to the 3' end and adapters were subsequently ligated onto the ends of the ds cDNA. Indices for multiplexing were attached during this ligation step. 13 cycles of PCR selectively amplified cDNA with adapter molecules on both ends. Using the molarity from Bioanalyzer (DNA 1000), libraries were diluted to 10nM. Libraries at 10nM were run on Bioanalyzer again (HighSensitivity DNA) and taking the lowest molarity, two samples with distinct barcodes were pooled. Clusters were generated using cBOT, and sequenced in one flow cell lane.