GSM8974249: NPM1 patient 08+09, diagnosis+relapse sample replicate 3,TotalSeq-A1-4 hash tag oligos; Homo sapiens; RNA-Seq
Cryopreserved AML samples were thawed at 37°C. Viable cells were enriched using an Annexin V MicroBead Kit (Miltenyi Biotec). After washing with cold PBS, cells were resuspended in 500 μl 0.5% PBA (0.5% BSA, 1 x PBS) with 0.2% human serum and incubated for 10 min on ice. After centrifugation and removal of the supernatant, cells were resuspended in 100 μl cold 0.5% PBA with 1 μl of unique cell hashing antibody (Biolegend, TotalSeq-A1-4) and incubated for 20 min on ice. Subsequently, cells were washed after spinning down and resuspended with an appropriate amount of 0.04% PBA (0.04% BSA, PBS) for the following procedures. Libraries were processed using a GemCode Single-Cell instrument and a Single Cell 3' Library & Gel Bead Kit v2 and Chip Kit (10x Genomics), according to the manufacturer's instructions, until the cDNA amplification step. Hashtag oligo (HTO) primers were added during the cDNA amplification PCR, and cDNA was generated following the 10x Single Cell 3' v2 protocol. Hashed cells were diluted to 1 million cells/ml and pooled together immediately before loading. Four samples (2 pairs) were pooled and loaded into one lane of a 10x Genomics Chromium Chip. Following PCR, 0.6X volume of SPRI beads were used to separate larger cDNA fragments (retained on beads) from cellular mRNA from the HTO-containing fraction (in supernatant). The cDNA fraction derived from cellular mRNA was processed according to the 10x Single Cell 3' v2 protocol. An additional 1.4X volume of SPRI beads was added to the HTO fraction to obtain a final SPRI volume of 2X SPRI. After cleanup and final elution, a separate PCR program was set up to generate HTO libraries.