title: GSM9268807: Stroma Control2+CHIP1+MDS3 multiplexed; Homo sapiens; RNA-Seq
library_construction_protocol: BM aspirates from a representative subset of donors in this study (3 age-matched controls, 3 CHIP, and 4 MDS donors), curated as part of the BoHemE study (NCT02867085), were included for scRNA-seq. The samples were thawed in 100% FCS + 100 µg/ml DNAse I, centrifuged at 300g for 5 min and suspended in DMEM + 10% FCS + 100 µg/ml DNAse I. When necessary, cell pellets were treated with 1X Red Blood Cell Lysis for 10 min at RT . Cells were washed with 2% FCS/PBS. Live/dead staining (FV780, Thermo Fisher) was performed in PBS, followed by antibody staining for 30 min at 4°C in CellCover (Anacyte Laboratories). Stained cells were sorted into PBS + 5% BSA with a FACSAria™ Fusion (BD Biosciences) equipped with a 130 µm nozzle into BSA-coated LoBind 1.5 ml tubes (Eppendorf). The HSPC and T cell fractions were combined and subjected to scRNA-seq in parallel with the stromal fraction, ensuring clean separation of the populations and minimizing contamination of blood or immune cells in the stromal fractions. 10x Genomics was performed using the Chromium Next GEM (Gel Bead-In Emulsions) Single Cell 3' kit (v3.1, 10x Genomics) according to the manufacturer's instructions. We pooled the T cells (CD45highCD34-CD14-CD235a-CD71-CD3+) and HSPC (CD45dimCD34+CD14-CD235a-CD71-) fractions in 1 lane and the stromal cells (CD34-CD45-CD14-CD235a-CD71-CD38-) in a separate lane. Sorting and single cell processing was performed in 3 batches on different days, with each batch consisting of a mix of control, CHIP, and MDS donors to correct for batch effects. Libraries were prepared based on manufacturer's instructions, with 13-14 PCR cycles for the final library generation. Following, the libraries were equimolarly pooled and sequenced using Illumina NextSeq 2000 with P2 and P3 flow cells.