title: GSM6736202: COH4 EXPT3 human CD34+ cells untreated (VEHICLE); Homo sapiens; RNA-Seq
library_construction_protocol: At the end of treatment, or after an additional 28 day treatment-free period, all mice were euthanised and the BM harvested from the two hind legs (ilia, femurs, and tibias) of each mouse by crushing in a mortar and pestle. BM was filtered and stained with mouse CD45-APC-Cy7, huCD45-PE, huCD34-APC and huCD38-Pe-Cy7 (all BD Biosciences) and the immunophenotypes of engrafted human cells determined by flow cytometry. For isolation of human cells from PDX model (huCD45+ and huCD45+34+) for scRNA-seq mouse BM cells were co-stained with muCD45 APC-Cy7 and huCD45 PE to distinguish mouse and human cells, together with huCD34 APC where appropriate, before being subject to FACS using the FACSAriaTM Fusion cell sorter (BD Biosciences) to isolate the specified human populations. Library was performed according to the manufacter's instructions (single cell 5' v1 protocol, 10x Genomics). Briefly, cells were resuspended in the master mix and loaded together with partitioning oil and gel beads into the chip to generate the gel bead-in-emulsion (GEM). The poly-A RNA from the cell lysate contained in every single GEM was retrotranscribed to cDNA, which contains an Ilumina R1 primer sequence, Unique Molecular Identifier (UMI) and the 10x Barcode. The pooled barcoded cDNA was then cleaned up with Silane DynaBeads, amplified by PCR and the appropriate sized fragments were selected with SPRIselect reagent for subsequent library construction. During the library construction Ilumina R2 primer sequence, paired-end constructs with P5 and P7 sequences and a sample index were added. Libraries were quantified using the Agilent Bioanalyzer High Sensitivity DNA Kit.