title: RNA-seq of mouse fetal liver endothelial cells, hepatoblasts and mesenchymal stromal cells at embryonic time points E13.5 and E17.5
library_construction_protocol: Female mice (6-12 weeks) were raised in groups of five and 72 hours before mating exposed to bedding material from a male cage. In the evening (after 5 p.m.) one female each was transferred to a cage containing a single housed male. The consecutive morning (before 9 a.m.) the breeding pair was separated, and females analyzed for the presence of a vaginal plug. That timepoint was designated as E0.5. Females were only mated for one night and 12 days later examined for a possible pregnancy. Embryos were extracted from the previously euthanized pregnant dams through a small abdominal incision and further dissected under a stereo microscope (Leica). Fetal livers were dissected using forceps and separated into single lobes using a surgical scalpel under a stereo microscope the lobes were fixed in 2 % paraformaldehyde (diluted in PBS) (6 h, 4 °C), washed twice in ice-cold PBS for five minutes and subsequently dehydrated in 30 % sucrose in PBS (24 – 48 h, 4 °C). The liver lobes were placed into base molds (15×15×5 mm) and completely covered in OCT medium, snap-frozen using liquid nitrogen and stored at -80 °C until use.  Dissected FLs were placed into 5 mL digestion medium (DMEM GlutaMAX, 10 mM HEPES, 10 % fetal bovine serum (FBS) in a 6-well cell culture dish, disintegrated by gentle pulling motions using two micro-forceps, collagenase (Type 2, 0.04 g/mL) and Deoxyribonuclease I (0.2 mg/mL) added and thoroughly resuspended using a 1 mL pipette. The cell suspension was immediately incubated for 45 minutes at 37 °C with gentle sample agitation. Following incubation, 5 mL of ice-cold calcium- and magnesium-free phosphate-buffered saline (PBS) containing 10 % FBS were added, the suspension filtered through a 70 μm cell strainer, centrifuged at 500 g (5 minutes, 4 °C), the supernatant discarded, and the pellet resuspended into a single cell solution in PBS.  Single cell suspensions were incubated with TruStain fcXTM (5 minutes, 4 °C) and subsequently immunostained using pre-conjugated antibodies (30 minutes, 4° C). All employed antibodies are listed in Table 1, including commercial sources. Next, the cells were washed twice in ice-cold PBS and resuspended in PBS containing DAPI (0.5 mg/mL), analyzed on a LSR II Fortessa (BD Biosciences). Post-acquisition data analysis was performed using FlowJo 10 software. Immunostained single cell solutions were prepared following the flow cytometry protocol explained above.    Phenotypically defined stromal cell populations were separated using a FACS Aria (BD Biosciences) and collected directly into RNase-free microfuge tubes containing 400 μL RLT lysis buffer (QIAGEN) supplemented with 4 μL β-mercaptoethanol and frozen down on dry ice for storage at - 80 °C. Between 6'000 and maximal 50'000 cells were collected depending on the abundance of each cell type. RNA was extracted from thawed cell lysates using the RNeasy Plus Micro Kit (QIAGEN) following the manufacturer's instructions and genomic DNA depleted using the supplied gDNA eliminator columns. RNA quality was judged by RIN values measured on a Bioanalyzer 2100 (Agilent, Waldbronn, Germany). Every analyzed sample displayed a RIN value above 9 Library preparation, cluster generation, and sequencing were performed at the functional genomics center Zurich (FGCZ, Zurich, Switzerland). The libraries were prepared using the SMART-Seq2 protocol (Picelli et al., 2014).