GSM6552954: Metaphysis, replicate 5, scRNAseq; Mus musculus; RNA-Seq
Cells of the bone microenvironment were isolated from the femurs of C57BL6/J mice. The femurs were separated into diaphysis and metaphysis with the epiphysis removed. Marrow cells were collected by flushing the diaphysis with PBS. Endosteal cells were isolated from marrow-depleted diaphyseal and metaphyseal bone fractions by gently crushing and cutting bones and digested in 2mg/ml of collagenase A and 2.5mg/ml of trypsin for 30 mins at 37ºC. After digestion, bone fractions were vortexed for 10s and the supernatant containing digested cells was filtered through a 100um filter into collection tubes containing 10% FCS. Marrow cells and endosteal cells were then centrifuged at 400 G for 5 mins. Cells of the bone microenvironment were isolated by FACS sorting by enriching for non-erythroid (Ter119-) and live cells (DAPI-). Libraries were prepared as according to the manufacturer's instructions (single cell 3' v2 protocol, 10x Genomics). Briefly, cells were resuspended in master mix before loading with partitioning oil and gel beads onto a 10X Chromium Next GEM Chip G to partition cells into GEMs (Gel Bead-In EMulsions). Gel beads were then dissolved to release primers (containing TruSeq Read 1 primer sequence, 10X Barcode and Unique Molecular Identifier, or UMI) and co-partitioned cells were lysed before incubating with reverse transcription reagents to generate barcoded, full-length cDNA. GEMs were then broken and the resulting cDNA pooled and purified using silane magnetic beads before amplifying via PCR. cDNA amplicon size was optimized by enzymatic fragmentation and subsequent fragment selection. Final libraries were generated by adding TruSeq Read 2 primer sequence, P5, P7 and a sample index via End Repair, A-tailing, Adaptor Ligation and PCR.