GSM9029842: Bone Marrow, E19 mice; Mus musculus; RNA-Seq
Fetal liver and spleen was collected, chopped and filtered through a 100 um strainer. Bone marrow was collected from the femur, tibia, pelvis, sternum and column through mechanical bone crushing. Cells were filtered through a 100um strainer and resuspended in PBS with 2% fetal bovine serum (FBS), followed by lysis of red blood cells using RBC lysis buffer (Biolegend, Catalog no. 420302). Cells were then enriched for c-Kit expression using CD117 MicroBeads (Miltenyi Biotec, Catalog no. 130-091-224). The resulting cKit-enriched fraction was washed and stained with fluorescently labeled antibodies: APC anti-mouse CD117, clone ACK2 (Biolegend, Catalog no. 105812), PE/Cy7 anti-mouse Ly6a (Sca-1) (Biolegend, Catalog no. 108114), Pacific Blue anti-mouse Lineage Cocktail (Biolegend, Catalog no. 133310) and PE anti-mouse CD201 (EPCR) (Biolegend, Catalog no. 141504). Cells were then washed and HSCs were purified through Flow Cytometry Cell Sorting using a CytoFLEX SRT (Beckman Coulter, Catalog no. C71883). Unperturbed P2 and E19 HSC isolation and sequencing were performed in different days and loaded on different reactions per tissue. Labeled P2 pups were stained and isolated as following: Pup1 BM - Totalseq #1, Pup2 BM - Totalseq #2, Pup1 Spl - Totalseq #3, Pup2 Spl - Totalseq #4. Then TdTomato+ from one pup was pooled with the TdTomato- of the other and viceversa: Tube 1 - 145 tdtomato positive from Pup1 (both BM and spleen mixed) + 5500 tdtomato negative from Pup2 (both BM and spleen mixed) and Tube 2 - 5156 tdtomato negative from Pup1 (both BM and spleen mixed) + 180 tdtomato positive from Pup2 (both BM and spleen mixed). These tubes were loaded into 2 single-cell reactions. Single-cell libraries using Chromium Single Cell 3' Reagent Kits (v3) following the manufacturer's guidelines (10X Genomics).