GSM8830800: CD34_2; Canis lupus familiaris; RNA-Seq
Bone marrow cells were collected from femurs that were stored on ice prior to processing. Single cell suspensions were collected by flushing the bone marrow cavity with sterile Hank's balanced salt solution and washed with phosphate-buffered saline (PBS). Red blood cells were then lysed using ammonium-chloride-potassium lysis buffer for 5-10 minutes at room temperature. The cell suspensions were again washed with PBS  and aliquoted for CD34+ cell enrichment and immunophenotyping. Viable (propidium iodide-negative) CD34+ cells were enriched from 2 of the 3 bone marrow samples (BM2 and BM3) using a Sony SH800 cell sorter, and post-sort purity was evaluated prior to GEM generation. For all three whole bone marrow samples prepared for GEM generation, viability was assessed using propidium iodide, and neutrophil proportions (CD4+CD3-CD5-CD18+ cells) were determined. Only samples with >70% viability and <25% neutrophils were used for sequencing. Sorted (CD34+ cells) and unsorted (whole bone marrow) were subject to a final 15-minute centrifugation (100 rcf at 8C), then cells were resuspended in 0.04% molecular grade bovine serum albumin (Sigma-Aldrich; St. Louis, MO) in PBS. Cell suspensions were adjusted to a concentration between 700-1200 cells/uL and were processed on a Chromium iX instrument (10x Genomics; Pleasanton, CA) within 30 minutes of preparation.  The instumnet was loaded to target 5,000 cells per sample. Library construction was performed according to the manufacturer's instructions (Chromium Next GEM Single Cell 3ʹ Kit v3.1 protocol, 10x Genomics). Briefly, a Chromium iX instrument was used to pass cells and oligonucleotide coated gel beads at a limiting dilution to obtain a coupling of cells and beads in a 1:1 ratio. The cell-bead pair was then passed from an aqueous phase to an oil phase to form an emulsion. Within the emulsions, cells were lysed, and the oligonucleotide sequences on the gel beads pull down message RNA and other RNA molecules that have long adenosine stretches with an oligo-d(T) pull down. After hybridization, reverse transcription is completed to generate a cDNA library with each transcript now associated with a unique cell barcode and an Illumina R1 primer sequence.  Once cells were barcoded and unique molecular identifiers (UMIs) added, a standard Illumina library preparation kit was completed with SPRIselect used for fragment size selection. During the library construction Ilumina R2 primer sequence, paired-end constructs with P5 and P7 sequences and a sample index were added.