GSM4983999: E17.5; Mus musculus; RNA-Seq
E15.5 and E17.5 embryos were isolated, and the skin, the brain, and some of the meningeal layers were peeled away, leaving the bony skull. For E15.5 embryos the coronal suture was dissected away from the skull cap using microdissection scissors, and 10 coronal sutures were rinsed in PBS and enzymatically dissociated with a final concentration of 3 mg/mL of Collagenase II (Worthington) and 4 units/mL of Dispase (COMPANY) in DMEM/F12 (company) for 45 min. For E17.5 embryos coronal sutures were dissected out in ice cold PBS using a scalpel blade, isolating a strip containing the overlapping frontal and parietal bone fronts (which appears opaque compared to adjacent regions) and avoiding the most apical and basal aspects of the suture. Isolated sutural strips from embryos from two litters in 3 batches (batch 1, 10 sutures from litter 1; batch 2 and 3, 3 sutures each from litter 2) were cut into small fragments in HBBS and digested using Collagenase IV (Worthington, USA; final concentration in HBBS of 2 mg/mL) for 30 min. Dissociation was terminated with 2% Fetal Bovine Serum and cells were passed through a 0.35 µM filter (E15.5) or Pluri-strainer Mini 70 µm (E17.5; pluriSelect Life Science, Germany). For E15.5 sample preparation, dead cells were removed using the Dead Cell Removal kit (Miltenyi Biotec 130-090-101) and cells counts were determined with a hemocytometer. The three batches of E17.5 dissociated cells were separately sorted by FACs to remove debris, cell doublets and likely dead cells (BD FACSAria Fusion; 100 µM nozzle) prior to library preparation. Transcriptome libraries  for single cells were captured using 10X genomics Chromium Single Cell 3' Library and Gel Bead Kit v2 following manufacturer's guidelines. Sequencing for E15.5 coronal sutures was performed with Illumina's HiSeq 3000/4000 PE Cluster Kit at the Children's Hospital Los Angeles' Molecular Genomics Core, and for E17.5 cells, PE sequencing was run on an Illumina HiSeq 4000 at the Oxford Genomics Centre Wellcome Centre for Human Genetics, Oxford achieving an average of  ~150,000 mean reads per cell for E15.5 and ~92,000 mean reads per cell for E17.5. scRNAseq