GSM8499467: S15-scDNA-seq_prot; Homo sapiens; OTHER
Mononuclear cells (MNCs) were isolated from each sample using the standard gradient separation approach with Ficoll-Paque PLUS (GE Healthcare Lifesciences, Marlborough, MA). According to the manufacturer's protocol, single-cell library DNA preparation was performed using the Tapestri Platform and reagents (MissionBio). Briefly, cryopreserved bone marrow mononuclear cells were thawed, washed with RPMI1640 supplemented with 40% FBS, 1% BSA, and 5 mM EDTA, and counted using a Countess cell counter (Invitrogen). BMNCs were normalized to 25,000 cells/μL in 40 μL and incubated with Human TruStain FcX (BioLegend, catalog# 422301), Blocking buffer in 1× staining buffer for 15 minutes on ice. Next, cells were stained with TotalSeq™-D Human Heme Oncology Cocktail (Biolegend, catalog# 399906), CD70 (Biolegend, catalog# 355127), CD99 (Biolegend, catalog# 371325), and CD366 (Biolegend, catalog# 345057) AOCs for 30 minutes on ice. Following staining, cells were washed three times in wash buffer and were loaded onto the Tapestri instrument for single-cell encapsulation, lysis, and barcoding with 2 μmol/L antibody tag forward primer. The DNA from barcoded cells was amplified via multiplex PCR using a targeted myeloid panel that included 279 amplicons across 37 genes associated with myeloid malignancies (https://designer.missionbio.com/catalogpanels/Myeloid-MDACC). DNA libraries were extracted from the droplets, followed by purification with Ampure XP beads (Beckman Coulter). The purified DNA libraries were indexed and then sequenced on Illumina's NovaSeq 6000 SP with 250-bp paired-end multiplexed runs. Protein PCR products, which exist in the supernatant from the Ampure XP bead purification step, were isolated by a 5-minute incubation with 2 μL 5′ biotin oligo for 5 minutes at 96°C, followed by a 5-minute incubation on ice. Isolated proteins were washed using 2× binding and washing buffer and streptavidin beads. Protein libraries were generated using the washed proteins, library template, and i5/i7 indices via PCR. The protein library PCR product was cleaned again using Ampure XP beads. Protein libraries were quantified, quality checked, and sequenced on Illumina's NovaSeq 6000 S4 with 150-bp paired-end multiplexed runs.