GSM7766520: Mouse_ID_53_57 wks DCA; Mus musculus; RNA-Seq
Measurements were taken from direct lysates of archived mouse liver sections derived from FFPE blocks. Two to three sections of 15 µM from each sample block were closely trimmed to remove excess paraffin and placed into -20°C storage until shipment overnight on dry ice to the vendor (BioSpyder, Inc., Carlsbad, CA) for processing. There, liver was transferred into lysis buffer, melted in mineral oil to remove remaining paraffin, and lysed. RNA within the lysate is annealed to a matched pair of detector oligos (DOs), with each pair of DOs targeting a single transcript within the mouse genome. Libraries were prepared as described in Trejo et al. (2019) (https://doi.org/10.1371/journal.pone.0212031). FFPE lysate was processed in annealing buffer with detector oligos (DOs), probes that have high selectivity for 2,205 Mus musculus target genes (TempO-Seq Mouse S1500+ surrogate attenuation panel, BioSpyder Technologies; Carlsbad, CA). After oligo ligation, a nuclease mixture was added to digest unbound or erroneously bound DOs. A final ligase mix was added to facilitate addition of a single primer pair. The ligases were deactivated with heat and the probes were amplified by PCR, which added sample specific barcodes that flank the target sequence and are inserted into the standard Illumina adaptors. PCR-amplified and barcoded samples were then pooled into a single library, purified and sequenced. TempO-Seq RNA samples were sequenced on the Illumina HiSeq 2500 platform with 50-bp single-end reads to a target read depth of 5 million. Overly abundant transcripts were attenuated equally across all samples to save sequencing reads.