title: GSM9083545: Bone, FibrousDysplasia2; Homo sapiens; RNA-Seq
library_name: GSM9083545
library_construction_protocol: Surgical bone was collected in 80% Dulbecco's Modified Eagle media (DMEM, Gibco) and 20% fetal bovine serum (FBS, Avantor, Seradigm) and transported on ice directly from the operating room to the laboratory for processing within 2–3 hours of surgical resection. Approximately 1 cubic centimeter of FD or WT bone was mechanically dissociated using a mortar and pestle for at least one minute until the bone fragments were white in color. Bone fragments were then enzymatically digested by incubating with type II collagenase (Gibco, 3000 U/ml) at 37⁰ C for 20 minutes with continuous gentle agitation and briefly vortexed every 5 minutes. The digested fragments were passed through a 70-μm prefilter, followed by a 40-μm filter, to collect the cells and strain the debris. Red blood cell lysis buffer (Biolegend) was added to the cells for 10 minutes in the dark, quenched with 1x PBS (Gibco), and followed by centrifugation at 600 x g for 5 minutes. Non-viable cells were removed using magnetic activated cell sorting (MACS) with a dead cell removal kit (Miltenyi Biotec). The resulting live cell fraction was enriched for CD45- cells using a MACS CD45 depletion kit (Miltenyi Biotec), following the manufacturer's instructions. Cells isolated after CD45 depletion were resuspended in PBS containing 0.04% bovine serum albumin (BSA) at a final concentration of 1,000 cells/μL. Library construction of 10x Genomics Single-cell libraries: Single-cell libraries were generated by the UC Davis Gene Expression Core 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. Library construction of GNAS amplified libraries: The remainder of the amplified 10x full-length cDNA library that was not used for 10X library generation was further amplified using standard Illumina sequencing primers (FOR-Illumina R1: 5′ CTA CAC GAC GCT CTT CCG ATC T-3′ and REV-Illumina template switch oligo primer: 5′-AAG CAG TGG TAT CAA CGC AGA GTA CAT-3′, Integrated DNA Technologies (IDT)). The amplified cDNA library contains full-length gene transcripts that are labeled with a 10X cell barcode (CB) and a unique molecular identifier (UMI). The GNAS transcripts were selectively amplified by PCR using the standard Read 1 Illumina sequencing primer (see above) and a second primer targeting exon 8 of GNAS (REV: 5′-CAA GAT CGA CGT GAT CAA GCA-3′). A customized partial Illumina Read 2 sequence was added to the 5′ end of exon 8 of GNAS via PCR (FOR Illumina R1, REV: 5′ GTT CAG ACG TGT GCT CTT CCG ATC TCA AGC AGG CTG ACT ATG TGC-3′), followed by the addition of P5 and P7 sample index (SI) Illumina adaptors (FOR-P5: 5′ REV-Partial Read 2-Sample Index – P7: 5′-CAA GCA GAA GAC GGC ATA CGA GAT N TGA CTG GAG TTC AGA CGT GT-3′ (where N= an eight–base pair individual sample index).
identifiers.namespace: GEO
identifiers.id: GSM9083545_r1