GSM9500443: mRNA E18.5 heart Lrrc58 hom Plagl1 mut(pat) rep 3; Mus musculus; RNA-Seq
Embryos from crosses between Lrrc58 homozygous trigger site mutant; Plagl1 wild-type dams and Lrrc58 heterozygous trigger site mutant; Plagl1 heterozygous trigger site sires were collected at E18.5 and dissected tissue was flash frozen in liquid nitrogen. Frozen tissue was transferred to a 50 mL conical tube, 1–2 mL of TRI Reagent was added, and the tissue was homogenized using a TissueRuptor and disposable probes (Qiagen). Following resuspension and/or homogenization, samples were phase separated with 100 μL 1-bromo-3-chloropropane (Sigma). RNA-seq libraries were prepared from total RNA using the Watchmaker RNA Library Prep Kit (Watchmaker Genomics). Ribosomal RNA depletion was performed using RiboDepletion Oligos (Qiagen) by mixing 14 μL of RNA input, 1 μL FastSelect reagent (Qiagen), and 10 μL Frag & Prime Buffer (Watchmaker Genomics) and incubating under the following conditions: 85°C 10 minutes, 75°C 2 minutes, 70°C 2 minutes, 65°C 2 minutes, 60°C 2 minutes, 55°C 2 minutes, 37°C 2 minutes, 25°C 2 minutes, 4°C. Samples were prepared from the First Strand Synthesis step of the Watchmaker RNA Library Prep Kit (Watchmaker Genomics) onward, according to the manufacturer's instructions. Libraries were multiplexed using xGen UDI Primers (IDT) and sequenced on the Illumina NovaSeq platform with paired-end reads.