title: GSM9012000: GC Ctx, replicate 2, scRNAseq; Mus musculus; RNA-Seq
library_construction_protocol: Femurs and tibia were dissected from tumor-bearing mice treated with either vehicle, GC Chemotherapy, GC Chemotherapy + Casp1i, or Casp1i for 2 cycles and kept on ice. Muscles were removed and bone marrow cells were collected by centrifugation at 13,000g for 15 seconds. Extracted bone marrow cells were treated with red blood cell removal buffer, for 5 min at room temperature. Lysis buffer was neutralized by the addition of 10X volumes of PBS and centrifuged at 300g at 4oC for 5 minutes. Centrifuged cells were resuspended in FACS buffer (0.5% BSA in PBS) and counted. Cells were then resuspended to a concentration of 10^7 cells/ml and hematopoietic progenitor cells were immuno-magnetically separated using the Hematopoietic Progenitor Isolation kit from Stem Cell Technologies following the manufacturer's instructions (Cat# 19856). Isolated hematopoietic progenitor cells were then counted and resuspended in FACS buffer to a concentration of 1000 cells/μL. Samples were finally processed according to the 10x Genomics protocol for the 3' v3.1 assay and were loaded to target 10,000 cell recovery. Library was performed according to the manufacter's instructions (single cell 3' v3.1 protocol, 10x Genomics). Briefly, hematopoetic progenitor 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 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 apropiated 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.