GSM5550363: 574_D; Bos taurus; RNA-Seq
PCLS from Plates 2 and 3 were collected after incubation, and transferred to separate screw-cap vials (490003-520, VWR, PA, USA), pre-filled with 600 µL of ice-cold TRIzol reagent (15596026, Thermo Scientific, MA, USA) and two 3.2 mm beads, homogenized using a Geno/Grinder Automated Tissue Homogenizer (2010-115, SPEX SamplePrep, NJ, USA; courtesy of the Department of Crop and Soil Sciences, Oregon State University, OR, USA), disrupting the tissue in short burst (45 seconds) at 1500 rpm, followed by incubation on ice for 3 minutes; the disruption-incubation cycle was repeated up to three times, or until no tissue pieces were visible within the tubes. Immediately after disruption, 120 µL of pre-chilled chloroform was added the tube, and the samples were mixed by inverting the tube, and incubated on ice for 5 minutes. After incubation, the samples were transferred to a new 1.7 mL microcentrifuge tube, and centrifuged a 4 C for 15 minutes, 15,000 x g. The upper-phase supernatant (~200 µL) was collected and RNA was purified using a Mag-MAX-96 Total RNA Isolation Kit (AM1830, Invitrogen, MA, USA) following the manufacturer's instructions, with minor modifications: briefly, 100 µL of each sample was transferred to the first row (A) of a 96-Well DeepWell Storage Plate (260251, Thermo Scientific, MA, USA). Rows B-F contained reagents supplied with the kit: 20 µL of the magnetic beads mix (row B), 150 µL of wash solution 1 (row C), 150 µL of wash solution 2 (row D), and 50 µL of DNAse-RNAse free water (VWRL0201-0500, VWR, PA, USA) in rows E and F. A suitable protocol was then generated to mimic the manufacturer's protocol with a KingFisher Duo Purification System (5400110, Thermo Scientific, MA, USA). Eluted RNA was measured using a SpectraDrop Micro-Volume Microplate in a SpectraMax plus 384 spectrophotomoter (89212-396, Molecular Devices, CA, USA). Average 260/230 and 260/280 ratios were 2.06 ± 0.24 and 1.69 ± 0.27, respectively. RNA integrity was assessed by the Center for Genome Research and Bioinformatics at Oregon State University using an Agilent Bioanalyzer 2100 (G2939BA, Agilent, CA, USA). For one animal, +10 DIM PCLS were excluded from the analysis, as their RIN was below 3; unsurprisingly, these samples clustered together in the principal component analysis of RNAseq results and were very dissimilar from the other animals, which further justified their removal; with that sample removed, resulting RNA integrity numbers were 7.26 ± 0.64. PCLS from postpartum animals treated with the three isotype-specific PPAR agonists, as well as the control group, were sent to the Center for Genome Research and Bioinformatics at Oregon State University for high-throughput sequencing. Library construction was obtained using a QuantSeq 3' mRNA-Seq Library Prep Kit FWD for Illumina (015.96, Lexogen, NH, USA). Sequencing was performed on an Illumina HiSeq3000 platform, at 60 samples/lane. The raw reads have been deposited