| Name |
Description |
Order Applied |
| GSM8384053 channel 1 growth |
Leukemia cells were not treated. |
1 |
| GSM8384053 channel 1 treatment |
Leukemia cells and organoids were co-cultured in special organoid growth media: Base: 1:1 (v/v) DMEM/F12 and Neurobasal Medium (Gibco). Supplements: 1:200 (v/v) N2 supplement, 1:100 (v/v) B27 supplement (w/o) vitamin A, 1:100 L-glutamine, 0.05 mM nonessential amino acids (MEM) (Gibco), 100 U/mL penicillin, 100 μg/mL streptomycin, 1.6 g/L insulin (Sigma-Aldrich), 0.05 mM β-mercaptoethanol (Gibco), ALK5 inhibitor (5 μM) SB431542 (Selleckchem) and the AMPK pathway inhibitor (0.5 μM) Dorsomorphin (Sigma-Aldrich). Coculture duration was 14 days |
1 |
| GSM8384053 channel 1 extraction |
Following the manufacturer's instructions, we used a Dynabeads™ CD19 Pan B (ThermoFisher) selection kit to isolate CD19+ cells from the organoid/leukemia cell suspension. The DETACHaBEAD CD19 kit (ThermoFisher) was used to separate beads from the suspension of positively isolated cells. RNA extraction was performed using the miRNeasy Micro Kit (50) #1071023 (Qiagen). The following cell quantities were isolated from 48 pooled organoids. For 697, we isolated 1.1 million cells from the organoids, and 0.4 million cells from the suspension. For Kasumi 2, we isolated 6.68 million from organoids, and 2.86 million cells from suspension. Flow cytometry analysis verified a 98% purity of CD19+ cells. Furthermore, automated cell counting indicated a viability exceeding 95% (Vi-Cell BLU, Beckman Coulter). Capillary gel electrophoresis was performed as a quality analysis and a 'Total RNA High Sensitivity Assay' (Agilent Technologies) on the FragmentAnalyzer System was implemented. The library preparation was performed according to the manufacturer's protocol using the 'Illumina Stranded Total RNA Prep, Ligation with Ribo-Zero Plus' (Illumina Inc.) kit. Briefly, the following steps were performed: rRNA depletion, fragmentation, cDNA synthesis, adapter ligation and library amplification. Bead-purified libraries were normalized and sequenced on the NextSeq2000 system (Illumina Inc.) with a read setup of SR 1x101 bp. |
1 |