title: GSM9162577: Experiment 24-022, dCas9/dCas12a system testing; Homo sapiens; RNA-Seq
library_construction_protocol: We performed flow cytometry and sorting using BD FACSMelody (100-µm nozzle) or BD Influx (140-µm nozzle) or BD FACSAria Fusion (100-140-µm nozzle) (BD Biosciences). We generated scRNA-seq libraries using the Smart-seq3xpress method and sequenced on a DNBSeq-G400RS platform (MGI, Shenzhen, China), as described in ref.26, using either HotMPS or StandardMPS chemistry. Smart-seq3xpress involves cell sorting by FACS into 384-well plates and downstream processing to achieve full-transcript coverage. Library construction protocol Exp23-001. At the end of the experiment (96 hr post-transfection), cells were dissociated and suspended in PBS containing DAPI (4 µg/ml) as a live/dead marker. FACS sorting was done using a BD Influx (﻿BD Biosciences), using a 140-um nozzle. Because transfected plasmids have largely been diluted out at this time point, we did not gate for expression of dCas9-mCherry-KRAB or dCas12a-miniVPR-GFP. This meant that many sorted cells were untransfected. During library preparation, we performed 12 cycles of pre-amplification PCR and 12 cycles of index PCR and used 0.002 ul TDE1 Tn5 enzyme per cell. Sequencing was performed using a SE100 sequencing kit (MGI). After sequencing and quality-control, the dataset consisted of 2959 cells. Library construction protocol Exp23-006. At the end of the experiment (72 hr post-transfection), cells were dissociated and suspended in PBS containing DAPI (4 µg/ml) as a live/dead marker. FACS sorting was done using a BD Influx (﻿BD Biosciences) using a 140-um nozzle, gating for dCas9-mCherry-KRAB expression and dCas12a-miniVPR-GFP expression. During library preparation, we performed 12 cycles of pre-amplification PCR and 12 cycles of index PCR and used 0.003 ul TDE1 Tn5 enzyme per cell. Sequencing was performed using a PE100 sequencing kit (MGI). After sequencing and quality-control, the dataset consisted of 1179 cells. We further computationally subsetted the dataset by including only cells that contained ≥1 read of any transfected construct, yielding a final dataset of 717 cells. Library construction protocol Exp24-010. At the end of the experiment (96 hr post-seeding), cells were dissociated and suspended in PBS containing DAPI (4 µg/ml) as a live/dead marker. FACS sorting was done using a BD Influx (﻿BD Biosciences) using a 140-um nozzle. During library preparation, we performed 12 cycles of pre-amplification PCR and 12 cycles of index PCR and used 0.003 ul TDE1 Tn5 enzyme per cell. Sequencing was performed using a PE100 sequencing kit (MGI). After sequencing and quality-control, the dataset consisted of 476 cells. Library construction protocol Exp24-019. Forty-eight hr post-transfection, cells were dissociated and suspended in PBS containing a live/dead marker (NucRed Dead 647; Thermo Fisher). FACS sorting was done using a ﻿BD FACS Melody (BD Biosciences) using a 100-um nozzle. Cells were sorted based on Lb-dCas12a-mCherry-KRAB expression and low fluorescence of the Live/Dead marker. During library preparation, we performed 12 cycles of pre-amplification PCR and 12 cycles of index PCR and used 0.003 ul TDE1 Tn5 enzyme per cell. Sequencing was performed using a PE100 sequencing kit (MGI). After sequencing and quality-control, the dataset consisted of 1334 cells. However, because our BD FACS Melody was not equipped with a UV laser, we had been unable to sort for cells expressing the As-dCas12a-VPR-BFP construct. We therefore subsetted the dataset to include only cells expressing high levels of the As-dCas12a-VPR-BFP (>100 reads per cell), yielding a final dataset of 543 cells. Library construction protocol Exp24-022. Forty-eight hr post-transfection, cells were dissociated and suspended in PBS. FACS sorting was done using a ﻿BD FACS Melody (BD Biosciences) using a 100-um nozzle. Cells were sorted based on dCas9-mCherry-KRAB expression and dCas12a-miniVPR-GFP expression. During library preparation, we performed 12 cycles of pre-amplification PCR and 12 cycles of index PCR and used 0.004 ul TDE1 Tn5 enzyme per cell. Sequencing was performed using a PE100 sequencing kit (MGI). After sequencing and quality-control, the dataset consisted of 1003 cells.