title: GSM8619063: Hashtags of samples from vehicle treated mice; Mus musculus; OTHER
library_construction_protocol: Tissue Harvesting Femur, tibiae, and spleen were harvested following euthanasia and submerged in SB5 buffer (phosphate-buffered saline (PBS) supplemented with 0.2% (w/v) bovine serum albumin (BSA), 0.08% (w/v) glucose, and 5 mM of Ethylenediaminetetraacetic acid (EDTA)). Tissue harvest methods are described in detail in Swaminathan et al.3. In brief, for bone marrow samples, the ends of the bones were cut and flushed with SB5 using a 26-gauge needle and 5 mL syringe. Harvested bone marrow was filtered through a 100 µM cell strainer. For spleen samples, spleens were placed onto a staining buffer-wetted 100 µM cell strainer and macerated through using the rubber side of a syringe plunger. The samples were washed with more SB5 and pelleted at 2000rpm for 10 minutes. Bone marrow and spleen samples were then processed into single-cell suspensions by adding 2 mL of SB5 and triturating cell pellet using a P1000 pipette and a large orifice pipet tip.  Singe cell RNA sequencing (scRNAseq) of bone marrow and spleen cells CD117 positive cell enrichment Harvested tissue samples were first enriched for CD117+ cells. Bone marrow and spleen cell samples were washed using Easy Sep buffer (PBS, 2% fetal bovine serum (FBS), and 1 mM EDTA) and enriched for CD117 expressing cells using EasySepTM Mouse CD117 Positive Selection Kit. The enrichment was performed largely following the manufacturer's protocol; however, all room temperature steps were instead performed at 4oC in a walk-in cold room.  Live cell enrichment using density centrifugation Following CD117 enrichment, dead cell and debris were removed using gradient centrifugation in OptiPrep. Solutions of 40%, 18%, 12%, and 5% OptiPrep were prepared using weight by volume (w/v) and pre-chilled. Cells were resuspended in 0.5 mL PBS and mixed with 1 mL of 40% OptiPrep in a 5 mL FACS tube. 18%, 12%, and 5% OptiPrep were sequentially and carefully overlay on-top of the cell sample. The samples were then spun at 800g for 15 minutes with the centrifuge break off to prevent disruption of gradient. The live cell layer, which formed between the 40% and 18% interface, was collected, and washed using cold PBS.  MULTIseq labeling of cell samples To reduce technical variability from sample processing, we performed MULTIseq labelling to multiplex samples for 10X Chromium-based encapsulation. Samples were labeled with MULTI-Seq Lipid-Modified Oligos and unique sample barcode oligos following the MULTI-Seq protocol (see "Sample_hashing_barcodes.csv" for MULTI-Seq barcodes per sample). Antibody-based hashing methods were tested for these cell samples but ultimately not used due to poor labeling of reticulocytes which lack of the hashing epitope. Single cell encapsulation and library prep Following MULTI-seq sample labeling, samples were pooled in equal ratios and cells were resuspended in PBS and 0.04% BSA at 1000 cells/uL prior to cell encapsulation. To help with demultiplexing, single injection condition pools were run individually, and an all-sample pool was run in another, for a total of five unique pools (see "Library_barcodes.csv") and a target total cell number of 48,000 loaded separately onto a 10X Genomics Chromium Next GEM Chip G. Samples were encapsulated using a Chromium X Instrument based on manufacturer's instructions.