Illumina NovaSeq 6000 paired end sequencing; Single cell RNA-sequencing of murine endothelial and stromal populations at embryonic day 17 (E17)
Single cell RNA-sequencing of murine endothelial and stromal populations at embryonic day 17 (E17)
To isolate stromal and endothelial cells from fetal bone, 9 fetuses were collected from a pregnant C57BL/6J mouse euthanized by cervical dislocation at day 17 of gestation (E17). Fetal long bones were dissected and cleaned using a dissection microscope. Collagenased bone fraction and bone marrow were obtained as previously described (Zambetti et al., 2016) and cell suspensions were stained using the following antibodies: anti-CD45.2 (clone 104, 1:100), anti-Ter119 (clone TER-119, 1:50), anti-CD51 (clone RMV-7, 1:50), anti-CD144 (clone BV13, 1:200), anti-Sca1 (clone D7, 1:100) all from Biolegend and  anti-CD31 (Clone  MEC 13.3, 1:100) from BD Biosciences. To discriminate between live and dead cells, staining with the viability dye 7-AAD (1:100) was performed. Endothelial cell enriched cell populations (7AAD- CD45- Ter119- CD31+) and stromal cells (7AAD- CD45- Ter119- CD31- Sca1+ CD51+) were FACS sorted. Sorted populations from bone marrow and bone fraction were pooled together and counted. An input of 17,000 cells was used for droplet based single cell RNA sequencing. Single cells were encapsulated for cDNA synthesis and barcoded using the Chromium Single Cell 3' Reagent Kit v3 (10X Genomics). Library construction was performed according to the manufacturerвЂ™s recommendations.