title: GSM7744880: T cell, Cas9 genome edited, MULTI-seq barcode; Homo sapiens; RNA-Seq
library_construction_protocol: Four days post-electroporation, T cells were labeled via MULTI-seq as previously described in McGinnis et al. Nat Methods (2019). Briefly, a lipid-modified oligonucleotide (LMO) was combined with a unique oligonucleotide barcode at a 1:1 molar ratio in PBS (−/−). 500,000 cells or fewer were washed twice with PBS (−/−) and then resuspended in PBS (−/−). The LMO/barcode solution was mixed with each cell suspension and incubated on ice for 5 minutes before addition of a co-anchor LMO and incubation on ice for an additional 5 minutes. Cold 1% BSA in PBS (−/−) was added to sequester free LMOs before washing cells twice with cold 1% BSA in PBS (−/−). Uniquely labeled cells were pooled in equal numbers in 1% BSA in PBS (−/−) to a final concentration of ~1,600 cells/mL. 10x Genomics Chromium Next GEM Single Cell 3' Gene Expression Kits (v3.1) were utilized to extract polyA RNA according to the manufacturer's instructions, with the following modification. Lanes of a standard Chromium chip were “super loaded” with ~50,000 cells to yield a target cell recovery of ~25,000 cells. scRNA-seq library construction was performed according to the 10x Genomics Chromium Next GEM Single Cell 3' Gene Expression Kit (v3.1) manufacturer's instructions, with the following modifications. During the cDNA amplification, 1 μL of a 2.5 μM MULTI-seq primer (see McGinnis et al.) was added. Supernatant from the cDNA bead cleanup was saved because it contained the MULTI-seq barcode amplicon. Supernatants were further cleaned by addition of SPRIselect beads and isopropanol with a conventional magnetic bead cleanup protocol. 3.5 ng of each cleaned amplicon was used in a PCR reaction to add sequencing indices; the reactions included KAPA HiFi HotStart ReadyMix (Roche), a unique i5 primer, and a unique RPI i7 primer. The PCR reactions were cleaned with SPRIselect beads before final library QC. The CROP-seq sgRNA was also enriched from the resulting cDNA according to Hill et al Nat Methods (2018). Briefly, 25 ng of cDNA was added to eight separate KAPA HiFi HotStart ReadyMix PCR reactions and amplified for 10 cycles with an annealing temperature of 65° C to enrich for the gRNA. Individual PCR reactions were pooled together and cleaned with SPRIselect beads. 8 µL of cleaned PCR1 product was added to a second KAPA HiFi HotStart ReadyMix PCR reaction and amplified for 10 cycles with an annealing temperature of 65° C to add Illumina sequencing adaptors. Gene expression, MULTI-seq barcode, and CROP-seq enrichment libraries were sequenced on an Illumina NovaSeq 600. Multiple iterations of library sequencing were concatenated to achieve the desired sequencing depth.