title: GSM8599466: organoid, CRISPRi, Cisplatin, CROP-seq; Homo sapiens; RNA-Seq
library_construction_protocol: The organoids infected with the sgRNA library were dissociated into single cells, and the live cells were FACS sorted (BD FACS Aria2) to isolate them for single-cell RNA-seq Cells were prepared for single-cell RNA-seq using the 10X Chromium Controller and Chromium Single Cell 3' Library & Gel Bead Kit v2 (10x Genomics). From the final library, the sgRNA sequences are specifically amplified with PCR, as described before, with the following primers: 5'-AATGATACGGCGACCACCGAGATCTACAC-3' and  5'-CAAGCAGAAGACGGCATACGAGATCAGCCTCGGTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGgtgttttgagactataaGtatcccttggagaaCCAcctTGTTG-3'. PCR cycling was performed according to the following protocol: (1) 95°C for 3 min, (2) 98°C for 15 s, then 70°C for 10 s (15 cycles) (3) 72 C for 1 min. The resulting sgRNA barcode library was purified via a 0.8X SPRI selection, and then further size was selected using BluePippin (Sage Science). sgRNA barcode libraries were sequenced as spike-ins alongside the parent RNA-seq libraries (5% spike-in) on a NovaSeq 6000, following the manufacturer's recommendations.