GSM8423250: Sequencing Pool 2,HTO; Homo sapiens; OTHER
Healthy and leukemic bone marrow samples were thawed using prewarmed RPMI with 10% FBS and cultured overnight. Subsequently, viable cells were subject to RNP-based CRISPR editting.  Cells were washed twice with cold PBS before labeling with unique TotalSeqA-compatible HashTags (BioLegend). CRISPR editted samples were incubated with human Fc receptor-blocking agent (BioLegend) and labeled with DAPI for live cell sorting by flow cytometry using an Aria cytometer (BD Biosciences). After sorting, equal numbers of cells were washed once with cold PBS before application of the TotalSeqA cocktail (BioLegend) and subsequently a custom ADT-tagged anti-CD10, CD90 and CD34 antibody (BioLegend). Cells were washed three times with cold PBS and submitted to the MSKCC Integrative Genomics Core for sequencing. A 10X Genomics Chromium machine was used for 3000–5000 single-cell capture. Cells were washed and partitioned into nanoliter-scale Gel Bead-In-Emulsions (GEMs), where all generated cDNA share a common 10x Genomics Barcode but uses a pool of ~750,000 barcodes to separately index each cell's transcriptome and ADT-barcode. The silane magnetic beads and Solid Phase Reversible Immobilization beads were used to clean up the GEM reaction mixture and the barcoded cDNA and antibodies was then amplified in a PCR step. Libraries were prepared using the Chromium Single Cell 3′ Reagent Kits (v3.1): Single Cell 3′ Library & Gel Bead Kit v3.1, Single Cell 3′ Chip G Kit v3.1 and Single Index kit T Set A (10x Genomics). Libraries were sequenced on an Illumina Nova Seq to achieve 75 bp reads. The P7 and R2 primers were added during the GEM incubation and the P5 and R1 during library construction via end repair, A-tailing, adapter ligation and PCR. The final libraries contain the P5 and P7 primers used in Illumina bridge amplification.