title: GSM9079268: AML-01-Dx-Blasts_GoT_NPM1_W288_runS35; Homo sapiens; OTHER
library_construction_protocol: Samples from AML-01, AML-02, AML-03, AML-04, AML-05, AML-06, CH-01, CH-02 were processed as follows. Frozen bone marrow and peripheral blood samples were thawed under standard procedures and prepared for sorting by flow cytometry. Briefly, cryopreserved samples were thawed and transferred to 15 ml conical tubes containing 10 ml of RPMI media with 10% fetal bovine serum (FBS) and 0.1mL of a 1 mg/mL DNAse I solution. Cells were centrifuged at 300 x g for 4 minutes at 4°C, and supernatant was removed. Cells were resuspended in FACS buffer and incubated with Fc blocker for 15 minutes. Next, cells were stained with PE-conjugated CD45, and FITC-conjugated CD3, CD19, and CD56 to enable lymphoid-depleted sorting. Cells were stained for 15 minutes and resuspended in FACS-DAPI to enable enrichment for live cells. Cells were then sorted for DAPI-, CD3-/CD19-/CD56- cells using the BD Influx at the Weill Cornell Medicine flow cytometry core. Sorted cells were processed using the 10X Genomics Chromium controller with the Single Cell Gene Expression 5' V1 protocol (except sample CH-05 which was processed on 5' V2 after V1 was discontinued). The 5' kit was selected to maximize genotyping efficiency because most mutations, including IDH1, IDH2, NRAS, and SRSF2, were closer to the 5' end of the mRNA. Single cell gene expression libraries were sequenced on a Nova-seq 6000 with R1:R2:I1 27:176:8bp paired end format where R1 contains the 10X barcode and UMI, R2 contains the transcript sequence, and I1 the sample index. Single cell genotyping was applied to 13 patients/donors (AML-01, AML-02, AML-03, AML-04, AML-05, AML-06, AML-16, AML-17, AML-18, AML-19, AML-20, CH-01, CH-02). Somatic mutations detected by clinical targeted DNA sequencing informed the design of multiplexed single cell genotyping using Genotyping of Transcriptomes (GoT).25 GoT targets were prioritized to capture mutations representative of distinct subclones with greater than 5% VAF in targeted DNA sequencing and sufficient gene expression in 10X data. Cells were profiled using the 5' 10X Genomics kit to maximize GoT efficiency, as most mutations were within 1000bp of the 5' end (IDH1/2, NRAS, NPM1, and SRSF2) (Figures S4B and S4C). GoT libraries were prepared for IDH1, IDH2, NRAS, NPM1, FLT3 and SRSF2 as described in Nam et al.25 We did not genotype DNMT3A which is often clonal and co-occurring within the same cells as IDH mutations, due to technical limitations of low gene expression (Average expression in single cell data: 0.19; comparable to IDH1 0.15) and >2000 base pair mutation distance from 5' end. Primers used for GoT are provided in Table S1. GoT Libraries were pooled and sequenced on Illumina MiniSeq 300 cycle mid-output kit in 147:8:147 paired end configuration or spiked in at 1-2% of gene expression library sequencing runs on NovaSeqX Plus with configuration as described above.