title: GSM7432172: Replicate 2, scRNAseq; Homo sapiens; RNA-Seq
library_construction_protocol: NP samples were obtained from patients with a diagnosis of IDD during surgery. NP samples from four patients in lumbar discectomy surgery were collected and minced with a razor blade into 1 mm3 fragments on a dish under aseptic conditions. NP fragments were suspended in 10 ml of 1×TrypLE Express and incubated in 37℃ water bath with shaking for 30 min. Centrifuge the tissue at 500g for 5 minutes at room temperature and wash twice by adding 25 ml PBS. Discard the supernatant and add 10 mL of digestion buffer consisting of 2 mg/mL Collagenase type II (Sigma, c6885), 0.1mg/ml DNase I (Roche, 4716728001) in RPMI medium (Coring, 10-040-CV), and incubated in 37℃ water bath with shaking for 4-6 hours. Terminate the enzymatic digestion when the tissue pieces have entirely disintegrated.  The suspension was passed through a 100 μm filter (Falcon, 3523260) and centrifuged (400g, 10 min, 4℃). Pelleted cells were resuspended in red blood cell lysis buffer (Solarbio, R1010) for 5 min at room temperature, followed by an additional filter step through a 40 μm filter (Falcon,3523240). Cells were collected by centrifugation (400g, 10 min, 4℃) and resuspended in PBS (BI, 02-024-1ACS) containing 0.04% BSA (Sigma, B2064).Cells were manually counted by Trypan blue (Thermo, T10282) and AO-PI (LUNA, D23001) after each centrifugation (400g, 10 min, 4℃) and resuspended. By using Chromium Next GEM Single Cell 3ʹ Kit v3.1 (10x Genomics, 1000268) and Chromium Next GEM Chip G Single Cell Kit (10xGenomics, 1000120), we performed single cell 3ʹ gene expression profiling. The cell suspension was loaded onto the Chromium single cell controller (10x Genomics) to generate single-cell gel beads in the emulsion according to the manufacturer's protocol. Captured cells were lysed and the released RNA were barcoded through reverse transcription in individual GEMs. Cell-barcoded 3ʹ gene expression libraries were sequenced on an Illumina NovaSeq6000 system by Shanghai Biochip Co., Ltd., Shanghai, China.