title: Transcriptomic profiling of BrO, DMGO and GD2 CAR T cells
design: Transcriptomic profiling of BrO, DMGO and GD2 CAR T cells
library_construction_protocol: Multiple organoids from the same batch and age were pooled. For timepoints after Matrigel embedding, Cell Recovery Solution (Corning, 354253) was used to dissolve the Matrigel. Organoids were incubated in this solution at 4°C for 15 minutes, halved, and washed in HBSS without Ca²⁺ and Mg²⁺. A single cell supsension was obtained using the Neural Tissue Dissociation Kit (Miltenyi Biotec, 130-092-628) according manufactures protocol.  Sequencing samples were thawed by warming the cryovials in a 37°C water bath until only a small clump of ice remained. The contents were then transferred to 10 mL of pre-warmed DMEM containing 10% FBS and centrifuged to pellet the cells. The cells were washed twice with PBS containing 5% BSA and filtered through a 40 µm Flowmi cell strainer to remove debris and aggregates. Approximately 30,000 dissociated cells per sample/timepoint were loaded into separate channels of a single Chromium controller (10x Genomics) chip. Consequently, gel bead-in-emulsions (GEMS) were generated using 10X Genomics Single Cell 3' Reagent Kit v4. Sequencing libraries were prepared using Single Cell 3' Reagent Kits v4 (10X Genomics) and then converted using the NovaSeq 6000 S2 Reagent Kit (Illumina). Library generation was provided by Leiden Genome Technology Center (Leiden, The Netherlands)