GSM2771310: 4a_90C_Con; Mus musculus; RNA-Seq
At the time of collection, liver lobes were systematically trimmed, and portions of left lateral, caudate, and right medial lobes were mixed, flash frozen in liquid nitrogen, and stored at -80°C. Frozen mouse liver samples were arbitrarily selected from control (Con) and PB groups (n=6/group), divided on dry ice into four portions (~20-30 mg each), and then preserved according to the following methods: stored at -80°C as frozen (FR); fixed in 70% ethanol for 3 months (OH); preserved in 10% buffered formalin for 18 hours followed by 70% ethanol for 3 months (18F); and fixed in 10% buffered formalin for 3 months (3F). Samples from OH, 18F, and 3F groups were kept at 4°C for 3 months, and then processed into paraffin blocks by standard histological methods: 80% ethanol for 30 minutes, twice; followed by 95% ethanol for 45 minutes each, twice; three changes of 100% ethanol for 45 minutes each; and three changes of xylenes (ThermoFisher Scientific, Fairlawn, NJ) for 30 minutes each; two changes of Paraplast™ Tissue Embedding Media (ThermoFisher Scientific, Fairlawn, NJ) for 30 minutes each and finally two changes of paraplast under vacuum for 1 hour each. The FR samples served as high-quality RNA controls. The 18-hour fixation time (18F) was selected to recapitulate a standard protocol for clinical and experimental samples, in particular those that may be used for immunohistochemical analyses (Fox et al., 1985). The 3-month formalin fixation (3F) was selected as an arbitrary long-term fixation scenario expected to have more severe formalin-induced RNA degradation. FFPE blocks were then stored at room temperature for up to 4 months until sectioning for RNA isolation. Total RNA was isolated from frozen (FR) liver samples following homogenization in RNAzolRT (Molecular Research Center, Cincinnati, OH) and then purified by RNeasy MinElute column according to manufacturer recommendations (Qiagen GmbH, Hilden, Germany). For FFPE samples (OH, 18F, and 3F), total RNA was isolated from two to four 10 µm-thick paraffin sections, which were collected with a Historange or Leica microtome under RNase-free conditions, deparaffinized (Qiagen Deparaffinization Solution, cat. #19093), and digested with proteinase K prior to RNA purification using the Qiagen AllPrep® DNA/RNA kit (Qiagen, cat. #80234), as described in Hester et al. (2016). To assess whether we could improve FFPE RNA quality for sequencing, additional sections from 3F were divided into demodification subgroups (DTAE, DQ, DP, and NoD). Total RNA was isolated from these samples using similar but slightly modified procedures to the protocol used for OH, 18F and 3F groups. DTAE RNA was isolated using the Qiagen AllPrep® DNA/RNA kit; however, after isolation, equal volumes of purified DTAE RNA and 2X TRIS-acetate-EDTA (TAE, final concentration 1X, pH 9.0; Sigma, St. Louis, MO) were combined and incubated for 30 minutes at 70°C. DQ RNA was isolated and purified using the Qiagen AllPrep® DNA/RNA kit according to the manufacturer’s protocol, except the 15-minute 80°C incubation following proteinase K digestion and RNA supernatant transfer was replaced by an incubation for ~18-hour at 55°C with 40 mM NaOH-buffered 2-amino-5-methylphenyl phosphonic acid (organocatalyst, final concentration 20 mM, pH 7.0; Evans Analytical Group, Maryland Heights, MO), which catalyzes the breakage of formaldehyde-induced aminal crosslinks and hemiaminal adducts on nucleic acids (Karmakar et al., 2015). DP RNA isolation was performed using the PureLink™ FFPE Total RNA Isolation Kit (Invitrogen, Carlsbad, CA; 92008, #K1560-02). FFPE sections were placed in paraffin melting buffer, digested in proteinase K, and incubated for ~18 hours at 55°C with 40 mM organocatalyst (final concentration 20 mM, pH 7.0). Following incubation, DP RNA purification proceeded as described in the manufacturer’s protocol. As a control for organocatalyst treatment, FFPE microtome sections for NoD were isolated exactly as for DQ, except that an equal volume of nuclease-free water (Ambion, Waltham, MA; cat. #AM9938) replaced the organocatalyst during the 18-hour incubation at 55°C. The concentration of RNA obtained from all samples was measured using a NanoDrop spectrophotometer (NanoDrop Technologies, Wilmington, DE) and Qubit fluorometer (ThermoFisher Scientific, Waltham, MA). RNA integrity was evaluated by an Agilent 2100 Bioanalyzer (Agilent Technologies GmbH, Berlin, Germany) prior to sequencing. Total RNA was converted into cDNA libraries using the Illumina TruSeq Stranded Total RNA with Ribo-Zero Gold Library Prep Kit (#RS-122-2303, Illumina, San Diego, CA). Briefly, ribosomal RNA (rRNA) was removed from 100 ng of total RNA by hybridization to a biotinylated probe selective for rRNA species, followed by streptavidin bead binding, purification, and fragmentation by heat in the presence of divalent cations. Double-stranded cDNA was generated by removing the RNA template and synthesizing the second strand with dUTP in place of dTTP. A single ‘A’ base was added to the 3’ end to facilitate ligation of sequencing adapters, which contained a single ‘T’ base overhang. Adapter-ligated cDNA was amplified by polymerase chain reaction (PCR) to increase the amount of sequence-ready library. During this amplification the polymerase stalls when it encounters a ‘U’ base, rendering the second strand a poor template. The first strand thus serves as template for amplified sequence, preserving strand information. Final cDNA libraries were analyzed for size distribution and integrity using an Agilent Bioanalyzer (DNA 1000 kit #5067-1504), quantitated by qPCR (KAPA Library Quant Kit #KK4824, KAPA Biosystems, Wilmington, MA), and normalized to 2 nM in preparation for sequencing.Samples were sequenced at EA Genomics (Q2 Solutions, Durham, NC) using the Illumina HiSeq platform
GEO Accession
GSM2771310