GSM9125225: TCF3::HLF B-ALL patient scRNA-seq (TAS-seq; IkawaWTA1); Homo sapiens; RNA-Seq
Cryopreserved bone marrow samples were thawed and incubated at 4°C for 10 min in Sulfo-NHS-LC-Biotin (10 μg/mL in PBS; EZ-Link™ Sulfo-NHS-LC-Biotin, Thermo Fisher Scientific, Cat# A39257) to biotinylate cell surface proteins. After three washes with Cell Staining Buffer (BioLegend, Cat# 420201), samples were incubated at 4°C for 15 min with TotalSeq™-A09xx PE-Streptavidin (corresponding to barcodes A0951: Patient1-Primary1, A0952:Patient1-Primary2, A0953:Patient1-Replase1, A0954:Patient1-Relapse2, A0955:Patient2-Primary1, A0961:Healthy donor1_Japan, A0962:Healthy donor2_Foreign country; final concentration 2.5 μg/mL; BioLegend) to label each sample with a unique DNA barcode. Cells were then stained with 1 μM Calcein AM at 37°C for 5 min to identify viable cells. Calcein(+) PE(+) 7-AAD(-) live cells were sorted using a FACS Aria III (BD Biosciences). Equal numbers of sorted cells from seven individual samples were pooled, and cells were loaded onto a BD Rhapsody cartridge (BD Biosciences, Cat# 665913) and processed using the BD Rhapsody Cartridge Reagent Kit (BD Biosciences), according to the manufacturer's instructions. Single cell suspensions were prepared from bone marrow samples and subjected to single-cell RNA preparation using BD Rhapsody. 10000 cells were loaded onto BD Rhapsody cartrige and captured cell number was asessed by BD Rhapsody scanner. Cell lysis, mRNA trap, cDNA synthesis, and primer digestion were performed according to the manufacture's instruction. cDNA amplification was performed by TAS-Seq protocol (Shichino et al. Commun Biol. 2022). Sequencing libraries from amplified cDNAs were produced by using NEB UltraII FS Kit for illumina (New England Biolabs) with some modifications. Sequencing was performed on an Illumina Novaseq 6000 sequencer at a depth of approximately 50000 reads per cell.