title: GSM8689833: MatsuiWTA1; Homo sapiens; RNA-Seq
library_construction_protocol: Mononuclear cells were obtained from freshly collected bone marrow cells. CD34-positive HSPCs were isolated using the EasySep Human CD34 Positive Selection Kit II (STEMCELL Technologies). Subsequently, 40,000 cells were immediately loaded into the BD Rhapsody Express Single-Cell Analysis System (BD Biosciences). scRNA-seq library was prepared using the TAS-Seq method (PMID: 35760847). Briefly, first-strand cDNA synthesis was performed on the BD Rhapsody Enhanced magnetic beads (BD Biosciences). Following exonuclease I treatment, a dC-tailing reaction was carried out at the 3' end of the cDNA using terminal deoxynucleotidyl transferase/RNase H enzymes. A mixture of dideoxycytidine 5′-triphosphate (ddCTP) and deoxycytidine triphosphate (dCTP) at a specific ratio was used to terminate the elongation reaction once ddCTP was incorporated. For the second-strand synthesis, a 5' universal-dG12 primer was employed instead of the 5' universal-dG9 primer described in the original protocol to enhance gene-detection sensitivity. After performing the first PCR with universal primers, size selection, and amplification via a second PCR, the library was ligated with Illumina TruSeq i7 adaptors (Illumina) and further amplified through PCR to add Illumina P5/P7 sequencing adapters.