GSM1788725: KO_ZT16_13016GP17; Mus musculus; RNA-Seq
Liver was extracted every four hours (0, 4, 8, 12, 16, and 20 h) and stored at -80C. Total RNA was extracted. Library construction was carried out using Illumina TruSeq Stranded mRNA Sample Prep Kit Set A. Samples were barcoded using kit adapters. 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, six samples with distinct barcodes were pooled. Clustering was carried out on the cBot using the TruSeq PE Cluster Kit v3-cBot-HS. Template DNA was prepared by diluting pooled libraries to 2 nM. The template DNA was denatured to single strands by mixing with NaOH. During this step, DNA is diluted to 20 pM. Denatured DNA was further diluted to desired pM input amount and spike in phiX. PhiX spike in varies by lane. Templates were loaded into an 8 strip tube which was loaded onto the cBot and protocol PE_Amp_Lin_Block_Hyb_v8.9 was used.