title: GSM8209376: Vehicle-d14, lin-cells, scRNAseq; Mus musculus; RNA-Seq
library_construction_protocol: Bone marrow samples obtained from recipients undergoing allogeneic hematopoietic stem cell transplantation (allo-HSCT) or from mice after allo-BMT transplantation were performed by density gradient centrifugation over Lymphoprep (07851, StemCell) to obtain BM mononuclear cells (BMMC). BMMC from VLA-4 A2- and vehicle-treated MDC mice at 2Ws and 5Ws after transplantation were further enriched through EasySep™ Mouse Progenitor Cell Isolation Kit (19856, StemCell) to obtain BM lineage negative cells. Single cell solution were kept on ice before loading to BD Rhapsody cartridge for single cell transcriptome capturation. Cells were firstly stained with two fluorescent dye, Calcein AM and Draq7, for precisely determination of cell concentration and viability via BD Rhapsody™ Scanner (BD Biosciences). Cells were loaded in one BD Rhapsody micro-well cartridge. Cell capture beads were then loaded excessively to ensure that nearly every micro-well contains one bead, and the excess beads were washed away from the cartridge. After lysing cells with lysis buffer, Cell capture beads were retrieved and washed prior to performing reverse transcription. Microbeads-captured single cell transcriptome were generated into cDNA library containing cell labels and unique molecular identifiers (UMI) information. All procedures were performed with BD Rhapsody cDNA Kit (BD Biosciences, Cat. No. 633773) and BD Rhapsody Targeted mRNA & AbSeq Amplification Kit (BD Biosciences, Cat No. 633801) strictly following the manufacturers' protocol. All the libraries were sequenced in a PE150 mode (Pair-End for 150bp read) on the NovaSeq platform (Illumina). For 10x Genomics, library was performed according to the manufacturer's instructions (single cell 3'v2 protocol). 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 retrotranscripted to cDNA, which contains an Illumina 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 appropriately sized fragments were selected with SPRIselect reagent for subsequent library construction.