title: Illumina NovaSeq 6000 paired end sequencing; Amplicon sequencing data from CROP-seq in 2 month old organoids
design: Amplicon sequencing data from CROP-seq in 2 month old organoids
library_construction_protocol: 409B2, a human induced pluripotent stem cell (iPSC) line derived from skin fibroblast, was used to create an iCRISPR-Cas9 nickase (Cas9n) and an iCRISPR-Cas9 line for CROP-seq experiment. The perturbation experiment was performed according to the CROP-seq protocol with some minor alterations. The experiment was performed in organoids derived from the inducible Cas9 nuclease line which contains a Puro selection marker. Cerebral organoids were generated using a whole brain organoid differentiation protocol (Lancaster et al. 2013, https://doi.org/10.1038/nature12517). The iCRISPR-Cas9 line was cultured on matrigel in mTesr1 supplemented with P/S (1:200) and Cas9 was induced 2 days prior to lentiviral transduction by adding 2µg/ml doxycycline. 24 hours later cells were dissociated into single cells with TrypLE and 300.000 cells of the iCRISPR-Cas9 cells were plated in at least 12 wells of matrigel-coated 6-well plates in mTesr1 supplemented with P/S (1:200), Y-27632 (final concentration 5µM) and 2µg/ml doxycycline. 24 hours later cells were transduced with a low multiplicity of infection (MOI) where less than 30% of the cells were GFP+ to ensure that the majority GFP+ cells only received one lentivirus per cells. The viral particles were added to the culture media (mTesr1 supplemented with P/S, Y-27632 and 2µg/ml doxycycline). 24h later, media was exchanged for mTesr1 supplemented with P/S and 2µg/ml doxycycline until 70% confluency was reached. Cells were then sorted for GFP-positive to enrich for CROP-seq-vector-positive cells and plated on matrigel-coated plates in mTesr1supplemented with 100 µg/ml Primocin (InvivoGen, ant-pm-1) and Y-27632 (final concentration 5µM). When cells reached 70% confluency, whole brain organoids were generated. After 2 months, single organoids and pools of multiple organoids were dissociated with a papain-based dissociation kit (MACS Miltenyi Biotech, cat. No. 130-092-628). Cells were sorted using fluorescence. Cell viability and number was assessed using the Trypan Blue assay and automated cell counter Countess (Thermo Fisher). Finally, cells were diluted to an appropriate concentration to obtain approximately 7,000 cells per lane of the 10x microfluidic chip. Single cell suspensions were acquired by dissociation of the organoids with a papain-based dissociation (Miltenyi Biotec, 130-092-628). Single cell RNAseq libraries were generated with the Chromium Single Cell 3' V3 Library & Gel Bead Kit. Single cell encapsulation and library preparation were performed according to the manufacturer's protocol.  gRNA were amplified from 60ng of cDNA remaining from scRNA-seq preparation with three separate PCR reactions. First, cDNA was amplified via PCR broadly targeting the outer part of the U6 promoter. Subsequently, the inner portion of the U6 promoter adjacent to the guide sequence and a TruSeq Illumina i5 adapter. Lastly, we added Illumina sequencing i7 adaptors. PCRs were monitored by qPCR to avoid overampliciation and following every PCR reaction the samples were purified using SPRI beads (Beckman Coulter).