GSM8031800: young lumbar disc; Mus musculus; RNA-Seq
Lumbar intervertebral disc 3 was carefully dissected from a 8-week-old mouse and then subjected to mechanical seperation and then collagenase digestion while incubating at 37 degrees Celcius for 1 hours . Following this, the solution underwent centrifugation, post which the supernatant was carefully discarded. The residual cell pellet was then resuspended in FACS buffer solution and live cells were isolated via Trypan blue staining. Library was performed according to the manufacturer's instructions (single cell 3' v2 protocol, 10x Genomics). Briefly, intervertebral disc 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 the 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 appropriate sized fragments were selected with SPRIselect reagent for subsequent library construction. During the library construction Illumina R2 primer sequence, pair-end constructs with P5 and P7 sequences and a sample index were added.