title: GSM6674677: Fusion_1; Mus musculus; RNA-Seq
library_construction_protocol: Total RNA from SCC7, Raw 264.7, and hybrid cells (n=3 per group) was isolated using TRIzol Reagent (Invitrogen Life Technologies), after which the concentration, quality, and integrity were determined using a NanoDrop spectrophotometer (Thermo Scientific). RNA sequencing libraries were prepared from 2 μg of total RNA with the following modifications: Ribosomal RNA was removed using an Epicenter Ribo-Zero rRNA Removal Kit (Human/Mouse/Rat). Fragmentation was performed using divalent cations at elevated temperatures in an Illumina proprietary fragmentation buffer. Using RNA as a template and random oligonucleotides as primers, the first strand of cDNA was synthesized and RNase H was used to degrade the RNA strand. In the DNA polymerase I system, dNTP with dUTP were used instead of dTTP as raw material to synthesize the second strand of cDNA. Double-stranded cDNA was purified. Double-end repair was then performed, the "A" base at the 3' end was introduced, and the sequencing adapter was connected. The USER enzyme (NEB, USA) was added to degrade the second strand of cDNA containing U. To select cDNA fragments of the preferred 400-500 bp in length, the library fragments were purified using the AMPure XP system (Beckman Coulter, Beverly, CA, USA). DNA fragments with ligated adaptor molecules at both ends were selectively enriched using the Illumina PCR Primer Cocktail in a 15 cycle PCR. The products were purified (AMPure XP system) and quantified using an Agilent high sensitivity DNA assay on a Bioanalyzer 2100 system (Agilent).