2 hrs post injection
The KAPA RNA Hyper Prep Kit (Illumina Platforms, Roche Sequencing Solutions, Pleasanton, CA) was used to generate the sequencing libraries following the manufacturer's protocols. Briefly, 1 g of mRNA was captured from total RNA using magnetic oligo-dT beads. Fragmentation was performed with a divalent cation (Mg2+) to the desired size by incubating at 85 C for 5 min using Fragment, Prime, and Elute Buffer (1). First and second strand cDNA synthesis was performed with random hexamers. After A tailing of cDNA fragments, 3-dTMP adapters are ligated to 3-dAMP library fragments using an adapter ligation master mix. Then, the ligated products were cleaned up with KAPA Pure beads. After post-ligation cleanup, the products were amplified by PCR using KAPA Library Amplification Master Mix with sequencing Primers and purified using KAPA beads to create cDNA library. Library fragment size distribution was confirmed by the Agilent Bioanalyzer 2100 system (Agilent Technologies, USA) and concentration was checked by Qubit 4 Fluorometer (Thermo Fisher Scientific, USA). The library was sequenced on an Illumina Novaseq 6000 platform, and low-quality reads were filtered out using internal software.