RNA-Seq of HT1 model mus musculus: adult mCdH-Heps_late passages
Total RNA concentrations were calculated using Quant-IT RiboGreen (Invitrogen, USA), and integrity values were accessed by the TapeStation RNA ScreenTape (Agilent Technologies, USA). Only high-quality RNA with an integrity number greater than 7.0 was used for library construction. A library was independently prepared with 1 g of total RNA for each sample using an Illumina TruSeq Stranded mRNA Sample Prep Kit (Illumina, Inc., San Diego, CA, USA). The initial step of library preparation involved purifying poly-A containing mRNA molecules with poly-T-attached magnetic beads. After this step, the purified mRNA was fragmented into small pieces using divalent cations under elevated temperature. The cleaved mRNA fragments were copied into first strand cDNA using SuperScript II reverse transcriptase (Invitrogen) and random primers, and the reverse complement strand of the cDNA was synthesized using DNA polymerase I, RNase H, and dUTP. These cDNA fragments then underwent an end repair process, which included the addition of a single A base and adapter ligation. After these steps, the final cDNA libraries were created, purified, and enriched with PCR. The libraries were quantified using KAPA Library Quantification kits for the Illumina Sequencing platforms according to the qPCR Quantification Protocol Guide (Kapa Biosystems, USA) and qualified by the TapeStation D1000 ScreenTape (Agilent Technologies). Indexed libraries were then paired-end sequenced with Illumina HiSeq 2500 (Illumina, Inc.) at Macrogen, Inc. (Korea).