GSM6628016: Liver, Carbon tetrachloride, 6 hours, Rep1; Mus musculus; RNA-Seq
For all time points, livers from three male C57BL/6J mice were harvested, flushed with PBS, immediately immersed in RNAlater (Qiagen), incubated at room temperature for 24 hours, and stored at -20 °C until future use. To isolate RNA, 30 mg of each tissue was removed from RNAlater and homogenized in 700 µL of QIAzol lysis reagent (Qiagen) using the TissueRuptor homogenizer (Qiagen). RNA was purified using the miRNeasy Kit (Qiagen) according to kit instructions and eluted in 30 µL of nuclease-free water. RNA sequencing libraries were prepared using KAPA mRNA HyperPrep Kit (KAPA Biosystems) according to manufacturer instructions. Briefly, 0.1-1 ug of total RNA was enriched for polyadenylated sequences using oligo-dT magnetic bead capture. The enriched mRNA fraction was then fragmented and first-strand cDNA generated using random primers. Strand specificity was achieved during second-strand cDNA synthesis by replacing dTTP with dUTP to quench the second strand during amplification. The resulting cDNA was A-tailed and ligated with indexed adapters. The library was amplified using a DNA polymerase that cannot incorporate past dUTPs to quench the second strand during PCR. The libraries were quantified using a KAPA qPCR Library Quantification Kit (KAPA Biosystems) as per manufacturer instructions.