Illumina NovaSeq 6000 paired end sequencing; Lineage recording in human cerebral organoids (whole organoids)
Whole organoids were dissociated for generating single cell suspensions by Papain based dissociation (Miltenyi Biotec, Neural tissue dissociation kit (P)). Single cells were encapsulated along with beads and reverse transcription reagents into droplets using 10X microfluidic chip system. Reverse transcription of first strand cDNA was performed in droplets. 2000 iTracer+ iCRISPR iPSCs per well in a 96-well plate were seeded and differentiated into cerebral organoids using a whole organoid differentiation protocol by Lancaster et al. (Nature Protocols, 2014). Throughout development organoids were scarred by activation of inducible Cas9. Scarring was achieved by first selecting the organoid to be scarred and transferring it to a 6-24-well plate (depending on organoid size) filled with 8ug doxycycline scarring media. Organoids were incubated in scarring media for 24 hours before returning to base media without doxycycline. Single stranded cDNA was cleaned up and amplified after breaking the droplets generated during the sample collection using the 10X 3' Single cell Kit according to the manufacturer's instructions. Quality and quantity of the resulting cDNA was assessed using Agilent's Bioanalyzer High Sensitivity DNA assay. Libraries were constructed following the 10X 3' library construction protocol. Briefly, cDNA was fragmented, end repaired and adaptor sequences were ligated. Barcode and scar regions were further amplified from 60-70ng of cDNA. Afterwards, a PCR was applied to introduce library specific sequencing primers as provided by 10X. Libraries were quantified and checked for size distribution using Agilent Bioanalyzer DNA High Sensitivity Assays.