title: GSM7101003: 4 day old; Mus musculus; RNA-Seq
library_construction_protocol: Mice were euthanized following the IACUC guidelines and sagittal, coronal, and lambdoid sutures were manually dissected using a dissecting microscope. Upon dissection, and consistently for all samples, no more than 1/4 mm of bone tissue surrounding the sutures was collected. Samples were incubated in 4 mL of collagenase solution (3.2mg Collagenase Type 2, Worthington-Biochem, catalog#LS004176, 1 mL of Trypsin-EDTA, GIBCO, catalog#25200-056 and 3mL of DPBS, GIBCO catalog#14190-144) for 20 minutes at 37°C, for 4 consecutive times. Cell suspensions derived from each incubation were pulled and centrifuged, and the obtained cell pellet was resuspended in 10mL of media. Library was performed according to the manufacter's instructions (single cell 3' v3 protocol, 10x Genomics). Briefly, cells were resuspended in the master mix and loaded together with partitioning oil and gel beads into the chip to generate the gel bead-in-emulsion (GEM). The poly-A RNA from the cell lysate contained in every single GEM was retrotranscripted to cDNA, which contains an Ilumina R1 primer sequence, Unique Molecular Identifier (UMI) and the 10x Barcode. The pooled barcoded cDNA was then cleaned up with Silane DynaBeads, amplified by PCR and the apropiated sized fragments were selected with SPRIselect reagent for subsequent library construction. During the library construction Ilumina R2 primer sequence, paired-end constructs with P5 and P7 sequences and a sample index were added. cDNA library QC and quantification were performed using Agilent 4150 TapeStation with High Sensitivity D1000 ScreenTape® Page.