GSM8656020: ligament, acl, acl23m; Homo sapiens; RNA-Seq
ACL samples were obtained during ACL reconstruction. All samples were transferred from the operating room on ice and stored in 30% FBS (fetal bovine serum, Gibco, #10437028) in Dulbecco's Modified Eagle Medium (DMEM) (Gibco, #11965084) until processing. Samples were then mechanically and enzymatically digested in 1 mg/ml collagenase III (Worthington Biochemical Corporation, #NC0021929) in DMEM with 10% FBS and 1% Penicillin/Streptomycin (Gibco, #15140122) at 37°C for 70 minutes with intermittent manual needle trituration with an 18-gauge needle. Digests were washed with PBS (Gibco, #10010023) and further digested with 0.25% trypsin-EDTA (Gibco, #25200056) at 37°C for 12–15 min. Suspensions were passed through 40 µm nylon mesh (Corning, #431750), erythrocytes were lysed with ACK lysing buffer (Quality Biological, #188-156-101) for 5–7 minutes on ice and washed in PBS with 2% FBS. Cells were then resuspended in 100 µl flow cytometry buffer (PBS with 2% FBS, 5 mM EDTA (Corning, #MT-46034CI), 2 mM HEPES (Quality Biological, #118-089-721)) per 1 gram of starting tissue weight. Cells were then frozen in 10% Dimethyl sulfoxide (DMSO, Sigma Aldrich, #276855) in FBS (fetal bovine serum, Gibco, #10437028) before flow cytometry and single cell sequencing. Cells were thawed, washed, and resuspended in flow cytometry buffer with 1:1000 SYTOX Blue (ThermoFisher, #S34857). Cells were resuspended in 0.04% BSA in PBS at 1000 cells per μl to prepare for scRNA-seq. Library was performed according to the manufacter's instructions (single cell 3' v2 protocol, 10x Genomics). Briefly, GCs 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.