GSM5462416: Single-cell RNA-seq assay (scRNA-seq) of the coronal suture at E18.5, replicate 1; Mus musculus; RNA-Seq
Coronal sutures, encompassing the SM, OFs, and frontal and parietal bones in the region of overlap, were dissected from C57BL/6J mice. Ectocranial and endocranial membranes were removed to the extent possible, and frontal and parietal bones were separated with forceps to expose the SM. Pooled E16.5 sutures were digested at 37ºC in αMEM (Gibco, 32571-036) with 0.2% collagenase type II (Worthington, LS004176), 0.2% dispase II (Sigma-Aldrich, 4942078001), and 1U/μl DNase (Qiagen, 79254). Sutures were serially digested with agitation five times for 10 minutes each in a shaking incubator. Successive fractions were pooled on ice with the addition of FBS to 2%. Cell suspensions were strained through a 40 μm filter (Falcon, 352340), pelleted at 400g for 7 minutes, washed in PBS/1% BSA, and resuspended in PBS/1% BSA. scRNA-seq 3' expression libraries were prepared on a Chromium instrument (10X Genomics, model GCG-SR-1) using the Chromium Single Cell Gene Expression kit (version 3 for E16.5 libraries; version 2 for E18.5 libraries).