title: GSM5048712: Sample5; Homo sapiens; RNA-Seq
library_construction_protocol: The nucleus pulposus tissues were surgically removed from patients and kept in MACS Tissue Storage Solution (Miltenyi Biotec) until processing. The tissue samples were processed as described below. Briefly, samples were first washed with phosphate-buffered saline (PBS), minced into small pieces (approximately 1mm3) on ice and enzymatically digested with 500 U/mL collagenase I (SangonBiotech) , 150 U/mL collagenase II (SangonBiotech) , 50U/mL collagenase IV (SangonBiotech) ,0.1mg/ml hyaluronidase (SangonBiotech),30U/mL DNaseI (SangonBiotech) and 5% Fetal Bovine Serum Oringin South America(Yeasen) for 95 min at 37°C, with agitation. After digestion, samples were sieved through a 70µm cell strainer, and centrifuged at 300g for 5 min. After washing with PBS containing 0.04% BSA, the cell pellets were re-suspended in PBS containing 0.04% BSA and re-filtered through a 35μm cell strainer. Dissociated single cells were then stained for viability assessment using Calcein-AM (Thermo Fisher Scientific) and Draq7 (BD Biosciences). The single-cell suspension was further enriched with a MACS dead cell removal kit (Miltenyi Biotec). Sequencing libraries were conducted using the BD Rhapsody Single-Cell Analysis System (BD Biosciences), following the manufacture's protocol. scRNA-seq