GSM9127839: FA161_female_56day_PFOA_0.166; Mus musculus; OTHER
Snap-frozen livers were cut on dry ice using a cooled scalpel. 12-25 mg of livers were homogenized in PowerBead tubes (Qiagen, cat#13114-50) containing 2.8mm ceramic beads and 600 μL of RLT buffer (from the RNeasy Mini Kit; Qiagen, cat#74104) supplemented with β-mercaptoethanol (10 μL/mL of RLT; Sigma-Aldrich, cat#M3148-25ML). Homogenization was carried out in a Qiagen PowerLyzer 24 Homogenizer (two 45-second cycles at 3500 rpm with a 30-second pause in between). Following homogenization, the lysate was transferred to a new tube and centrifuged at 13,000xg for 2 minutes. The supernatant (lysate) was transferred to a new tube, and ribonucleic acid (RNA) was extracted with Qiagen RNeasy Mini Kit (Qiagen, cat#74104) according to the manufacturer's instructions. TempO-Seq mouse S1500+ surrogate gene panel (version 1.2; BioSpyder Technologies Inc.) was used to generate sequencing libraries to measure gene expression in total RNA purified from mouse liver tissues. Each RNA sample was first diluted to 100 ng/mL in water, then mixed with an equal volume of 2× TempO-Seq Lysis buffer, resulting in a final RNA concentration of 50 ng/µL. Two microliters of this diluted mixture (approximately 100 ng of RNA) were used as input for TempO-Seq library building. As per the manufacturer's protocol, RNA was first hybridized with the detection oligos (DOs). Unbound DOs were digested with a nuclease, and the remaining bound DOs were ligated. These ligated oligos were then amplified by PCR using primers with sequencing tags to create the sequencing libraries. The resulting libraries were pooled together and quantified using qPCR with the KAPA SYBR FAST Universal kit, compatible with Illumina platforms (Roche, Wilmington, MA). The pooled libraries were sequenced using a 75-cycle flow cell on an Illumina NextSeq 500.