Illumina NovaSeq 6000 sequencing; Lineage recording in human cerebral organoids (iTracer-perturb)
Whole organoids were dissociated for generating single cell suspensions by Papain based dissociation (Miltenyi Biotec, Neural tissue dissociation kit (P)). RFP+, GFP+, and BFP+ cells were sorted into separate pools using FACS. 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. RFP/GFP+ (iTracer-TSC2-targeting iCRISPR) and BFP/GFP+ (iTracer-non-targeting) cells were sorted into separate FACS tubes and plated in 12-cell matrigel-coated plates with mTeSR1. Following cell recovery after FACS, 2000 cells per well in a 96-well plate (10% RFP/GFP+, 10% BFP/GFP+, and 80% wildtype) were seeded and differentiated into cerebral organoids using a whole organoid differentiation protocol by Lancaster et al. (Nature Protocols, 2014). Organoids were scarred by activation of inducible Cas9 at day 5. Scarring was achieved using 8ug doxycycline in NIM for 24 hours. 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 v3 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 v3. 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.