GSM7978121: Day 2, Replicate 2, 10X; Homo sapiens; RNA-Seq
We produced a pool of lentiviruses containing the CROP-seq sgRNA library and transduced NPC with relatively low titer of lentiviruses (10-20% transduction efficiency) to minimize multiple infections in which a cell receives more than one sgRNA. NPC cultures after puromycin selection were used for the CROP-seq experiment. At day 0, day 2 and day 4 of differentiation, cells were harvested using papain (Worthington, Cat#9001-73-4). Cells were prepared according to 10x Genomics general sample preparation protocol.At each time point, ~90k cells were loaded into 3 wells (~30k cells per well) of the Chromium Next GEM Chip G for GEM preparation. GEMs of each time point were stored in -20°C. Single cell library preps of all 9 samples from the three time points were conducted together according to the protocol provided for Chromium Next GEM Single Cell 3' Reagent Kit v3.1 (10x Genomics, Cat#PN-1000121). Concentrations of libraries were measured using Agilent High Sensitivity DNA kit (Agilent Technologies, Cat#5067-4626) on Bioanalyzer. At each time point, ~90k cells were loaded into 3 wells (~30k cells per well) of the Chromium Next GEM Chip G for GEM preparation. GEMs of each time point were stored in -20°C. Single cell library preps of all 9 samples from the three time points were conducted together according to the protocol provided for Chromium Next GEM Single Cell 3' Reagent Kit v3.1 (10x Genomics, Cat#PN-1000121). Concentrations of libraries were measured using Agilent High Sensitivity DNA kit (Agilent Technologies, Cat#5067-4626) on Bioanalyzer. Briefly, three PCR reactions were performed followed by 1x SPRIselect beads (Fisher Scientific, Cat#NC0406407) cleanup after each PCR reaction. In PCR1, 15ng full-length cDNA from single cell RNA-seq library prep per sample was used as a template. In PCR2, 10ng post-cleanup PCR1 product was used as a template. In PCR3, 10ng post-cleanup PCR2 product was used as a template to add sample indices. All PCR reactions were conducted using KAPA Hotstart HiFi ReadMix (VWR, Cat#103568-584) with annealing temperature at 62°C (15 sec) and extension at 72°C (15 sec) for 18 cycles (PCR1) or 15 cycles (PCR2 and PCR3). Concentrations of post-PCR3 products were measured using Qubit dsDNA HS assay kit (Thermo Scientific, Cat#Q32854). Samples of single cell RNA-seq libraries and enriched sgRNAs were pooled together at 5nM for sequencing on S2 and S4 flow cells of Illumina NovaSeq. Approximately 1 billion reads were generated per sample.