title: GSM8549497: Chimeroid cells, TSHZ3 shRNA-treated and mCherry-, single-nuclei multi-ome (gene expression); Homo sapiens; RNA-Seq
library_name: GSM8549497
library_construction_protocol: For PIP-sequencing: Chimeroids were harvested at week 8 for single-cell sequencing using the PIPseq™ T2 3' Single Cell RNA Kit v4.0PLUS (Fluent Biosciences). Three chimeroids from the single-shRNA conditions and 33 chimeroids from the double knock-down conditions were prepared for flow-assisted cell sorting as described above for the organoid knock-down experiments. Target populations were sorted on a FACSAria (BD Biosciences) configured with a 561-nm laser, 595LP dichroic mirror, and 605/40-nm bandpass filter for mCherry detection; a 481-nm laser, 505LP dichroic mirror, and a 530/30-nm bandpass filter for GFP detection; and a 355-nm laser, 450 LP dichroic mirror, and 515/30-nm bandpass filter for DAPI detection. Sorted samples were pelleted and resuspended in 2% FBS in PBS to obtain an estimated 5,000 cells / µL. This enabled the targeting of ∼ 25,000 cells for capture. To maximize our ability to obtain high-quality single-cell profiles, we conducted at least two PIP captures for each sorted cell population. For 10x multi-ome: Chimeroids were harvested at week 10 for single-cell multi-omic profiling using Chromium Next GEM Single Cell Multiome ATAC + Gene Expression Kit CG000338 Rev F (10X Genomics). Eight chimeroids from the FEZF2-shRNA and nine chimeroids TSHZ3-shRNA conditions were prepared for flow-assisted cell sorting as described above, with over 100,000 cells obtained per sort. Nuclei were extracted as per the 10X Nuclei Isolation Protocol (CG000365), using a 3-minute incubation in lysis buffer and 3, 1-mL washes with Wash Buffer. For PIP-sequencing: Sequencing libraries were obtained from cells according to PIPseq T2 instructions, using 16 cycles for cDNA amplification and 8 – 12 cycles for library amplification depending on the amount of DNA. For 10x multi-ome: Sequencing libraries were obtained from cells according to manufacturer instructions, using 7-9 cycles for ATAC library amplification; 6-9 cycles for cDNA amplification; and 14 – 16 cycles for gene expression library amplification depending on the amount of DNA. All libraries were quantified on an Agilent 2100 Bioanalyzer, and sequenced as per manufacturer recommendation on a NovaSeq X Plus flow cell. scRNA-seq; single-nuclei multi-ome (gene expression and ATAC)