Illumina NovaSeq 6000 paired end sequencing; Lineage recording in human cerebral organoids (Visium)
Organoids were embedded in pre-chilled Optimal Cutting Temperature (OCT). The sample was then set into a dry ice bath with isopentane until frozen and stored at -80°C. Cryosections were cut at 10um thickness, adhered to ST slides (10x) and stored at -80°C until the following day. Tissue slices were fixed in cold methanol, before being stained in Hematoxylin and eosin. ST slides were imaged as recommended on a Nikon T2i at 20x using a tile scan over all slice sections. 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). Organoids were scarred by activation of inducible Cas9 at day 15. 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. Tissue slices were permeabilized to obtain RNA, optimal permeabilization conditions were determined by using the Tissue Optimization Kit (10x) and was found to be 24 minutes. Spot-captured RNA was reverse transcribed before second strand synthesis and cDNA denaturation. qPCR was used to determine the optimal number of cDNA amplification cycles as recommended by the manufacturer. cDNA was amplified using 17-18 cycles. Libraries were constructed following the 10X Visium library construction protocol v1. 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.