Single-cell sequencing of acute myeloid leukemia sample
Single cell sequencing was performed by using Mission Bio's Tapestri AML platform, which assesses hotspot mutations in human AML, according to the manufacturer's protocol. Briefly, cryopreserved bone marrow aspirates or peripheral blood mononuclear cells were thawed and counted before loading apprixiately 150 000 cells onto the Tapestri microfluidic cartridge. Cells were emulsified with lysis reagent and incubated at 50 degrees C before thermally inactivating the protease. The emulsion containing the lysates from protease-treated single cells was then microfluidically combined with targeted gene-specific primers, PCR reagents, and hydrogel beads carrying cell-identifying molecular barcodes using the Tapestri instrument and cartridge. After generation of this second, PCR-ready emulsion, molecular barcodes were released in a photocleavable manner from the hydrogels with UV exposure, and the emulsion was thermocycled to incorporate the barcode identifiers into amplified DNA from the targeted genomic loci. The emulsions were then broken by using perfluoro-1-octanol, and the aqueous fraction was diluted in water and collected for DNA purification with SPRI beads (Beckman Coulter). Sample indexes and Illumina adaptor sequences were then added via a 10-cycle PCR reaction, and the amplified material then underwent SPRI purification a second time. After the second PCR and SPRI purification, full-length amplicons were ready for quantification and sequencing. Libraries were analyzed on a DNA 1000 assay chip with a Bioanalyzer (Agilent Technologies) and sequenced on an Illumina MiSeq with 150 bp paired-end chemistry. A single sequencing run was performed for each barcoded single-cell library prepared with our microfluidic workflow. A 5% ratio of PhiX DNA was used in the sequencing runs. See Ediriwickrema A, Aleshin A, Reiter JG, et al. Single-cell mutational profiling enhances the clinical evaluation of AML MRD. Blood Adv. 2020;4(5):943-952.
NCI-phs003853
SU290