GSM2772992: D267ALI14; Homo sapiens; RNA-Seq
Cells were incubated with 0.10% protease type XIV from Streptomyces griseus in HBSS for 4 hours at 4°C degrees. Cells were  detached from the Transwells by pipetting and then transferred to a microtube. 50 units of DNase I per 250µl, were directly added and cells were further incubated at room temperature for 10 min. Cells were washed in 500 µL HBSS 10% Fetal Bovine Serum then in 500 µL HBSS before being mechanically dissociated through a 26G syringe (4 times). Finally, cell suspensions were filtered through a Scienceware® Flowmi™ Cell Strainer (40µm porosity), then centrifuged (150g for 5 min) and resuspended in 500 µL of cold HBSS. Cell concentration measurements were performed and 1500 cells were used for single-cell sequencing. Single cell RNA-Seq libraries were made following the User Guide for v2 Chemistry (https://support.10xgenomics.com/single-cell/sample-prep/doc/user-guide-chromium-single-cell-3-reagent-kit-v2-chemistry). Library was sequenced as follows: 26bp Read 1 (16bp Chromium barcode and 10bp UMI), 57bp Read 2 (transcript), and 8bp i7 sample barcode for each scRNA-Seq library.