title: GSM7318411: Endochondral bone, E18.5, scRNAseq; Mus musculus; RNA-Seq
library_construction_protocol: Forelimb long bones (humerus, radius and ulna) were dissected from fetuses at embryonic day E18.5 and pups at postnatal day 1 and carefully cleaned of surrounding tissue.  Cell suspensions for sorting were prepared according to Houlihan et al., 2012 (PMID 23154782) with the following specific modifications. Bones were cut in small pieces with a scalpel and all tissue was processed (bone marrow cells were not washed out or flushed) for enzymatic digestion using collagenase D (2mg/ml in DMEM [high glucose]).  Red cell lysis was not performed. The time of digestion was 45-50 minutes in a water bath at 37ºC with 3 rounds of gentle pipetting every 10 min to help disaggregation. Collagenase digestion was stopped in ice and by addition of ice-cold 10%FBS/HBSS+ (HBSS, 10mM HEPES, 1% Penicillin-Streptomycin). Cells in suspension were recovered to a new tube and any remaining bone pieces were transferred to a ceramic mortar and 2 steps of very gentle tapping with the pestle (20-30 times) was applied to increase cell recovery. Cells in the mortar were recovered with the addition of 10%FBS/HBSS+, filtered through a 100µm strainer and pooled with the cell suspension set aside.  After centrifugation (350g for 10 min at 4°C), cells were resuspended in 2%FBS/HBSS+ for FACS. Before antibody staining, blocking of FcγR II/III was performed by incubation in ice with anti-CD16/CD32 antibodies for 15 min (1mg  per million cells). The gates were defined by the use of a mix of pan-antibodies to mesenchymal (CD140a/PDGFR-a) and endothelial cells (CD31/PECAM), together with CD9, a marker highly expressed in early hematopoietic progenitors and stromal cells. Collection tubes for sorting were precoated for 15 minutes with 10% FBS/HBSS+ in ice, and kept at 4º C during sorting.  Samples were analyzed and sorted in a SONY MA900 (lasers: 488/561 and 405/638nm). 7AAD (7-amino actinomycin D) was the only dye used to discriminate dead cells.  Dead cells and multiplets were excluded by gating, and no lineage labeling was used to prevent the loss of any cell subsets. The following four gates were used: CD9hi, PDGFRa/CD31hi; CD9hi, PDGFRa/CD31low; CD9low, PDGFRa/CD31hi; CD9low, PDGFRa/CD31low and sorted cells were mixed using a 10:1:1:2 ratio to represent both abundant and minor populations and processed for scRNAseq. scRNAseq libraries were constructed according to the manufacter's protocol (Chromium Next GEM Single Cell 3' Kit v3.1; 10x Genomics). Cells suspensions representing all heterogeneity within the perinatal endochondral bone 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 DynaBeads, amplified by PCR and the apropiated sized fragments were selected with SPRIselect reagent for subsequent library construction. P5, P7, i7 and i5 sample indexes, and TruSeq Read 2 (read 2 primer sequence) are added via End Repair, A-tailing, Adaptor Ligation, and PCR. The final libraries contain the P5 and P7 primers used in Illumina amplification.