GSM7012094: maternal lung, TiO2, 12.11 h, replicate 1; Mus musculus; RNA-Seq
Isolation of pups and maternal/fetal organs: At 2, 5, 12 and 24 h, dams were terminally anesthetized by subcutaneous injection of 0.2 ml of Zoletil mixture (tiletamin/zolazepam, xylazin og fentanyl) and killed by exanguination by withdrawal of heart blood into Eppendorf tubes containing 36 ml K2EDTA (n=7-9 per exposure/time point). The uterus was excised and opened. Fetuses were excised from their embryonic sac, their viability confirmed, killed by decapitation, sexed by visual inspection, and their position in the uterus noted. From each litter, the first female fetus encountered in the right uterine horn, counting from the cervix, was selected and saved for analyses. The placenta was dissected into chorion and decidua by blunt/stump dissection under. From dams, the liver and right lung were dissected. Dissected organs were snap frozen in liquid nitrogen and kept at -80ºC. Fetal livers were dissected later. Maternal blood was centrifuged at 2000xg at 4°C for 5 min and plasma was stored in aliquots at -80°C until analysis. A maximum of one female fetus from each dam were used for any one outcome. RNA extraction: Total RNA was isolated from frozen maternal lung and liver, chorion, decidua and fetal liver. Briefly, 20 - 50 mg of tissue was homogenized with a blender in 700 ul lysis buffer with 7 ul mercapto-ethanol. RNA extraction was carried out utilizing magnetic beads technology, on a chemagic Prepito® (Perkin Elmer, Waltham, Massachusetts), as recommended by the manufacturer. Concentration and purity were measured on a NanoDrop1000 spectrophotometer, with all samples showing an A260/280 ratio between 1.9 and 2.1. RNA integrity was analyzed by 2100 Bioanalyzer (Agilent Technologies) with Agilent RNA 6000 Pico Kit (Agilent Technologies) as recommended by manufacturer. All samples used for RNAseq displayed RNA integrity number (RIN) above 7. cDNA library construction and paired end sequencing was carried out by Novogene (Hongkong). To exclude ribosomal RNA, polyA selection was done. Novogene paid service