Perturbational single-cell RNA-seq time course of CD34+ HSPC megakaryocyte differentiation with CMO multiplexing (Days 0–7, 1 uM compounds)

We profiled transcriptomic responses of a panel of small molecules during in vitro megakaryocyte (Mk) differentiation of CD34+ hematopoietic progenitors from a single donor. Cells were cultured in SFEM with TPO and CC100; an untreated Day 0 sample (unhashed) was collected, and additional samples on Days 1, 2, 5, and 7 were multiplexed with Cell Multiplexing Oligos (CMOs) and pooled 12 samples per library (~100k cells per well contributed), with duplicates for each compound distributed across libraries and plate-level positive/negative controls. Libraries were prepared with 10x Genomics Single Cell 3’ v3.1 and sequenced on Illumina NovaSeq 6000. FASTQs were generated with bcl2fastq and processed with Cell Ranger (STAR/GRCh38). Hashed data were QC-filtered, demultiplexed by Gaussian mixture modeling of CLR-normalized CMO counts, and further filtered to singlets; counts were then normalized to 10,000 per cell and log-transformed. For consistent annotation, the dataset was mapped to a hematopoiesis reference using harmony-corrected PCs and SymphonyPy KNN label transfer, with robustness assessed by distance/similarity metrics and marker expression. This submission provides Day 0 raw counts, Days 1–7 raw counts (pre-demultiplexing), demultiplexed singlet counts with well/treatment assignments, and normalized counts with transferred cell-type labels.