GSM7523803: Wild Type Sham2; Mus musculus; RNA-Seq
Frozen PLT muscles were pulverized into a fine powder using an RNase-treated and liquid nitrogen-chilled mortar and pestle. The resulting powder for each sample was collected, weighed, and placed in 0.5 ml of ice-cold TRIzol. Samples were then homogenized with a Polytron (PT 1200 E) for 20 s and then centrifuged at 6,000 x G for 1 min to remove bubbles and confirm complete homogenization. Total RNA was isolated with a PureLink RNA Mini Kit according to the manufacturer's instructions. The purity and integrity of RNA samples were checked by the ratio of A260/A280 and of 28S/18S rRNA, respectively, and then confirmed by the University of Wisconsin-Madison Biotechnology Center using the NanoDrop One Spectrophotometer and Agilent 2100 Bioanalyzer. Total RNA libraries were prepared from samples that met the TruSeq® Stranded Total RNA Sample Preparation Guide (15031048 E) input guidelines using the Illumina® TruSeq® Stranded Total (Gold) RNA Sample Preparation kit. For each library preparation, cytoplasmic & mitochondrial ribosomal RNA was removed using biotinylated target-specific oligos combined with paramagnetic beads tagged with streptavidin. Following purification, the reduced RNA was fragmented using divalent cations under elevated temperature. Fragmented RNA was copied into first-stranded cDNA using SuperScript II Reverse Transcriptase and random primers. Second-strand cDNA was synthesized using a modified dNTP mix (dTTP replaced with dUTP), DNA Polymerase I, and RNase H. Double-stranded cDNA was cleaned up with AMPure XP Beads (1.8X). The cDNA products were incubated with Klenow DNA Polymerase to add a single 'A' nucleotide to the 3' end of the blunt DNA fragments. Unique dual indexes (UDI) were ligated to the DNA fragments and cleaned up with two rounds of AMPure XP beads (0.8X). Adapter ligated DNA was amplified by PCR and cleaned up with AMPure XP beads (0.8X). Final libraries were assessed for size and quantity using an Agilent DNA1000 chip and Qubit® dsDNA HS Assay Kit, respectively. Libraries were standardized to 2 nM. Paired-end 150 bp sequencing was performed, using standard SBS chemistry on an Illumina NovaSeq6000 sequencer.