GSM8776994: MDS41_01_CD45Neg; Homo sapiens; RNA-Seq
Bone marrow aspirates were obtained from MDS/AML patients after written informed consent in accordance with the Declaration of Helsinki and approval of University of Rochester institutional review board (IRB). To isolate the bone spicules, the bone marrow aspirates were passed through 40mM filter. The filter containing spicules was washed with PBS and placed in a 6 well plate. The spicules were digested for 1 h in collagenase (Stem cell technologies) at 37°C. After filtering, the filtered aspirate was used to isolate bone marrow mononuclear cells (BMMNCs) by density centrifugation (Ficoll-Paque, GE healthcare). Digested bone cells and BMMNCs were magnetically depleted for CD45- cell fraction using LD columns (Miltenyi Biotech). Cell suspensions were processed to generate single-cell RNA-Seq libraries using Chromium Next GEM Single Cell 3′ GEM, Library and Gel Bead Kit v3.1 (10x Genomics), per the manufacturer's recommendations. Samples were loaded on a Chromium Single-Cell Instrument (10x Genomics, Pleasanton, CA, USA) to generate single-cell GEMs (Gel Bead-in-Emulsions). GEM reverse transcription (GEM-RT) was performed to produce a barcoded, full-length cDNA from poly-adenylated mRNA. After incubation, GEMs were broken, the pooled GEM-RT reaction mixtures were recovered, and cDNA was purified with silane magnetic beads (DynaBeads MyOne Silane Beads, ThermoFisher). The purified cDNA was further amplified by PCR to generate sufficient material for library construction. Enzymatic fragmentation and size selection was used to optimize the cDNA amplicon size and indexed sequencing libraries were constructed by end repair, A-tailing, adaptor ligation, and PCR. Final libraries contain the P5 and P7 priming sites used in Illumina bridge amplification. Sequence data was generated using Illumina's NovaSeq 6000. Samples were demultiplexed and counted using cellranger-8.0.0 mkfastq and count, using default parameters.