title: GSM8060211: DAOY-cerebellar organoid, coculture, replicate 1; Homo sapiens; RNA-Seq
library_construction_protocol: Day 60 organoids and organoid-tumour co-culture samples were collected using a wide bore pipette tip and placed in a 6 well plate. Using micro-scissors, organoids were cut in four pieces before placing a total of five organoids in an Eppendorf tube. After removal of excess medium, 200 uL of pre-warmed (to 37°C) Neuron isolation enzyme was added and the tube was placed in a 37°C water bath. Organoids were incubated with the enzyme for 30 minutes with intermittent agitation. The enzyme was removed and organoids were washed once with HHGN medium (HBSS + 2.5mM HEPES + 35mM Glucose + 4mM NaHCO3). Organoids were then dissociated in differentiation medium by gentle trituration. Afterwards, the cell suspension was enriched for viable cells using the dead cell removal kit (Miltenyi Biotec) following the manufacturers protocol. Cell viability was determined using Trypan Blue staining and counted using an automated cell counter (Applied Biosystems Countess 3). Cells were centrifuged at 300 x g for 5 minutes and resuspended in the appropriate volume of resuspension buffer (HBSS + 0.04% BSA) to a final concentration of 2 x 10^3 cells / uL before proceeding with single cell RNA sequencing. Thirty tumour spheroids were collected using a wide bore pipette tip and placed in an Eppendorf tube. Excess medium was removed and 200 uL of pre-warmed Neuron isolation enzyme (Gibco) was added. The tube was placed in a 37°C water bath for 20 minutes and agitated every 5 minutes. Subsequent processing was performed following the same steps as described above. DAOY and ONS-76 cell monolayer cultures were dissociated by incubation with pre-warmed TrypLE for 5 minutes. TrypLE was then diluted in PBS before centrifugation at 400 x g for 5 minutes to collect the cells. Cells were washed once in differentiation medium before processing as described above. Single cell barcoding and reverse transcription of poly-A mRNA was performed using the 10X Chromium controller (3' version 3.1 chemistry) and sequencing of single cell libraries was performed on an Illumina NovaSeq6000 sequencing system according to the manufacturer's instructions