GSM5709571: Neonatal IVD 1; Homo sapiens; RNA-Seq
The IVDs were dissected, nucleus pulposus identified and washed with Phosphate Buffered Saline (PBS). The tissue was manually minced and enzymatically digested. Tissue was digested with Pronase Protease (Millipore, Temecula, CA) in growth media, followed by ~18 hour digestion of Collagenase Type 1S in growth media. The resulting sample was pushed through a 70μm cell strainer and cells were isolated via centrifugation. The pellet was resuspended in phosphate buffered saline at a concentration of 1,500 cells/μL and 100uL of sample was processed for single-cell RNA sequencing. Chromium Single-cell 3' v2 libraries were prepared on a Chromium Controller with chips and reagents from Single-cell Gene Expression v2 kits following the manufacturer's protocols (10x Genomics). The libraries were then sequenced using paired-end sequencing on an Illumina NovaSeq. Samples were sequenced with raw sequencing data analyzed and visualized with pre-release versions of Cell Ranger 3.0.0 and Loupe Cell Browser 3.0.0. Raw sequencing data was demultiplexed and converted to fastq format by using bcl2fastq v2.20 (Illumina, San Diego, CA). Fragment analysis of indexed libraries was performed on the Agilent 4200 TapeStation (Agilent Technologies, Santa Clara, CA).