Chapter 4 138 Following mechanical trituration, enzymatic dissociation was performed using the Papain Dissociation System (Worthington Biochemical Corporation, LK003150) following the manufacturer’s instructions, with minor modifications. Briefly, the minced spheroids were incubated with papain and transferred to an orbital shaker at 70 rpm inside a tissue culture incubator for 70 minutes. To stop the reaction, dissociated fragments were pelleted and resuspend with a papain inhibitor. After being resuspended in 0.2 % bovine serum albumin diluted in PBS supplemented with 10uM Y-27632, cells were filtered through a 70 µm cell strainer to remove any undissociated tissue clumps. Cell counting was performed using TC20 automated cell counter (BioRad), and viability was calculated as the percentage of live cells (trypan blue-negative) over the total cell count. Viability for each individual sample was evaluated to ensure it was above 90%. Finally, cells were suspended in sterile PBS 0.04% BSA to a final concentration of 1 × 106 cells/mL (1,000 cells/μl). For single cell sequencing approximately 6400 cells were loaded onto a single-cell chip for GEM generation using the 10x Genomics Chromium Controller (10× Genomics, Pleasanton, CA), to obtain a target output of 4000 cells per sample. 3′mRNA-seq gene expression libraries were constructed using the Chromium Single Cell 3′ Library & Gel Bead Kit v2 (10x Genomics) according to the manufacturer’s guidelines. All reactions were performed in a C1000 Touch Thermal Cycler (Bio-Rad Laboratories, Hercules, CA). Twelve cycles were used for cDNA amplification, while the number of total cycles for the sample index PCR was calculated based on the cDNA concentration. Amplified cDNA and final libraries were evaluated using a Tape Station 4200 (Agilent Technologies, Santa Clara, CA) with a high sensitivity chip. scRNA-seq libraries were sequenced using an Illumina NovaSeq6000 with the standard sequencing protocol of R1 28; I1 10; I2 10; R2 90 nt read length to obtain 50.000 reads per cell. Single cell encapsulation, cDNA amplification, library prep and sequencing were performed at COSR (Center for Omic Sciences). Bioinformatics Analysis of the scRNA-seq data was performed by the Bioinformatics core of the San Raffaele Telethon Institute for Gene Therapy according to previously described methods (Naldini et al., 2023).
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