GSM5710228: GTE009; Homo sapiens; RNA-Seq
Fresh EPN tissue was excised by physicians from Beijing Tiantan Hospital and delivered on ice to Institute of Genetics and Developmental Biology of Chinese Academy of Sciences immediately. Microdissected tissues were transferred to a 24-well cell culture plate and digested by buffer comprising 20 U/mL Papain (Worthington, LK003178), 100 U/mL DNaseI (Worthington, LK003172), 10 U/L chondroitinase ABC (Sigma, C3667), 0.07% hyaluronidase (Rhawn, R006687), 1 XGlutamax (Life Technologies, 35050061), 0.05 mM (2R)-amino-5-phosphonovaleric acid (APV; Thermo Fisher Scientific, 010510), 0.01 mM Y27632 dihydrochloride (Sigma, T9531), and 0.2 XB27 supplement (Thermo Fisher Biosciences, 17504044) in Hibernate-E media (Life Technologies, A1247601) for 1-2 hrs at 37°C, and then pooled with Hibernate-E buffer containing 1×Glutamax, 0.05 mM APV, 0.2X B27, 0.01 mM Y27632 dihydrochloride. Tissues were gently triturated through Pasteur pipettes with finely-polished tips of 600, 300 and 200 μm diameters in order, and washed once with Hibernate-E buffer to generate single-cell suspensions. After filtration through a 40 μm strainer (Thermo Fisher Scientific, 130-101-812), 1 X red blood cell lysis solution (MACS Miltenyi Biotec, 130-094-183) was added to remove blood contamination during surgery followed by 1800 μL debris removal solution (MACS Miltenyi Biotec, 130-109-398). Subsequently, the dissociated cells were stained with DAPI (0.2 µg/mL) to identify dead cells. scRNA-seq libraries were constructed under the manufacturer instructions provided by 10×Genomics accompanying single cell 3' Library and Gel Bead Kit V3 (10×Genomics, 1000075). A Chromium Single Cell Controller (10×Genomics) loaded cell suspensions (300-600 living cells per microliter determined by Count Star) to generate single-cell gel beads in the emulsion (GEM). Quality control was performed on the generated cDNA library using the Agilent 4200 and scRNA-seq was performed on the Illumina Novaseq6000 sequencer.