GSM8242742: D7-rep2_GEX; Mus musculus; RNA-Seq
Following euthanasia, bone marrow was harvested from the femur, tibia, pelvis, and sternum through mechanical crushing, ensuring the retrieval of most HSCs. The collected bone marrow cells were then sieved through a 100-μm strainer and cleansed with a cold 'Easy Sep' buffer containing PBS with 2% fetal bovine serum (FBS), followed by lysis of red blood cells using RBC lysis buffer (Biolegend, Catalog no. 420302). At first, mature lineage cells were selectively depleted through the Lineage Cell Depletion Kit, mouse (Miltenyi Biotec, Catalog no. 130-110-470), while the resulting Lin- (lineage-negative) fraction was then enriched for c-Kit expression using CD117 MicroBeads (Miltenyi Biotec, Catalog no: 130-091-224). These cKit-enriched cells were washed, blocked with FcX and stained with following 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); PE anti-mouse CD201 (EPCR) (Biolegend, catalog no. 141504); PE/Cy5 anti-mouse CD150 (SLAM) (Biolegend, catalog no. 115912); APC/Cyanine7 anti-mouse CD48 (Biolegend, catalog no. 103432). For scRNA sequencing and subsequent plating, cells were pipetted up and down gently a few times to be dissociated into single cells and transferred to a 1.5 mL microtube. The well was then washed with prewarmed PBS to collect all the possible remaining cells. Cells were then concentrated by centrifugation at 800 g for 8 minutes. Washed cells were then blocked with FcX, and stained with the E-SLAM stem cell antibodies panel, to confirm expansion of E-SLAM cells. Live cells were then sorted based on fluorescent reporter expression. Part of the sample as specified in text was then taken for constructing a single-cell library using Chromium Single Cell 3' Reagent Kits (v3) following the manufacturer's guidelines (10X Genomics). The remaining part was then plated back for further expansion in culture. To minimize the impact of batch effects on sequencing, we multiplexed different conditions leveraging the unique barcode pattern of our libraries together with Biolegend TotalSeq™ anti-mouse hashing antibodies, enabling the simultaneous preparation of libraries representing all experimental conditions in a single reaction for each day of sampling.