title: GSM7235331: diseased annulus fibrosus (Thompson grade III-IV) single cell RNAseq, donor5; Homo sapiens; RNA-Seq
library_construction_protocol: Spine samples from postmortem donors with no recorded history of spine trauma or disc degeneration were obtained from Lifesharing under IRB approval. Lumbar spines were resected with surrounding muscles intact, wrapped in sterile gauze and transported on ice. The specimens were washed with 70% EtOH and the discs were isolated intact using an Orthopedic bone saw OTS-1 (Orthopedic Drills & Medical Devices, Covina, USA). IVDs were cut to expose the center and macroscopic grading was performed by applying the Thompson grading system. Using a sterile scalpel, the AF and NP tissues were isolated. The inner transition zone between the NP and AF was removed to prevent mixing of the cells from the two compartments. AF and NP were separately stored on ice in sterile DPBS (Gibco #14190144) containing 10% calf-serum. Under sterile conditions the isolated NP and AF tissues were washed in 70% EtOH, weighed, and homogenized into very fine pieces for enzymatic digestion and single cell isolation. Single cell RNA-seq was performed using the 10X Genomics Chromium technology. Briefly, cells were partitioned into droplets using the Chromium Controller with the Next GEM Single Cell 3' Reagent Kit v3.1 using dual indexing. Next, cells were lysed and the polyadenylated transcripts reverse transcribed to generate barcoded cDNA following the recommended 10X Genomics protocol. The quality of both the cDNA and the final libraries were assessed using the Agilent Bioanalyzer 2100 following the guidance provided by the 10X Genomics user guide. Libraries were loaded onto 100-cycle flowcells for sequencing using the NextSeq2000 (Ilumina) and sequenced as follows: read1 = 28 cycles, i7 index = 10 cycles, i5 index = 10 cycles, read 2 = 90 cycles. 10,000 cells were targeted per sample with a target of 20,000-25,000 reads per cell.