Sample 17_s
The dams were euthanized 18.5 days post coitum by cervical dislocation. Whole brain, intestine and placenta were collected from two fetuses per dam, to a total of 12 fetuses per experimental group. The fetal organ samples were immediately frozen in liquid nitrogen, stored at − 80 °C and transferred on dry ice to the research laboratory. Six pregnant germ-free (GF) and six specific pathogen free (SPF) C57BL/6 J dams were obtained from the EMMA Axenic Service at Instituto Gulbenkian de Ciência, Portugal. The GF dams were 3–4 months old and the SPF dams 4–5 months old. The GF and SPF statuses were regularly monitored by culture and 16S qPCR. All dams were fed identical RM3-A-P (Rat and Mouse No.3 Breeding Autoclavable) breeding diets (SDS Special Diet Services, Essex, UK), autoclaved at 121 °C. The SPF feed was autoclaved for 20 min and the GF feed for 30 min due to logistical reasons. Total RNA was extracted from fetal intestine, brain and placenta (one female fetus and one male fetus per dam) using the Qiagen RNeasy Mini Kit (Qiagen, Germany). The tissues were mechanically lysed by grinding with a plastic pestle (Bel-Art, PA) attached to an electric drill, and the protocol included the optional on-column DNAse digestion. Dual-indexed mRNA libraries were prepared from 150 ng of total RNA with QuantSeq 3' mRNA-Seq Library Prep Kit FWD (Lexogen Gmbh, Vienna, Austria) according to user guide version 015UG009V0251. During second strand synthesis, 6 bp Unique Molecular Identifiers (UMI) were introduced with UMI Second Strand Synthesis Module (Lexogen Gmbh, Vienna, Austria) for detection and removal of PCR duplicates. Quality of the libraries was measured with LabChip GX Touch HT DNA High Sensitivity Reagent Kit (PerkinElmer, Waltham, MA, USA).