title: GSM9478903: Cerebral cortex, E15, Mock Infection, Mouse Litter 2, Replicate 2; Mus musculus; RNA-Seq
library_construction_protocol: For bulk RNA-sequencing of the brain and placenta, pregnant dams were euthanized via cervical dislocation. Embryos were harvested from the dams and stored in ice-cold HBSS over ice as the placentas and embryonic brains were dissected in ice-cold HBSS. The placentas and dorsal cortex of offspring were dissected, placed in RNAProtect overnight, and  transferred to -20°C for later archival storage. To extract the RNA from placenta and dorsal cortices, tissue were homogenized using Lysis Matrix D ceramic beads. After centrifugation of the homogenate, the supernatant was extracted and total RNA was purified via the Qiagen RNeasy Mini kit. RNA samples were submitted to Yale's Center for Genome Analysis for cDNA library preparation (Kapa mRNA HyperPrep) and sequenced (NovaSeq6000) at 100 million reads per sample. RNA Seq Library Prep: mRNA is purified from a normalized input between 50-1000ng of total RNA with oligo-dT beads and sheared by incubation at 94C in the presence of Mg (Kapa mRNA Hyper Prep). Following first-strand synthesis with random primers, second strand synthesis and A-tailing are performed with dUTP for generating strand-specific sequencing libraries. Adapter ligation with 3' dTMP overhangs are ligated to library insert fragments. Library amplification amplifies fragments carrying the appropriate adapter sequences at both ends. Strands marked with dUTP are not amplified. Indexed libraries that meet appropriate cut-offs for both are quantified by qRT-PCR using a commercially available kit (KAPA Biosystems) and insert size distribution determined with the LabChip GX or Agilent TapeStation. Samples with a yield of ≥0.5 ng/ul are used for sequencing.