GSM7101908: MPP1_GEX_Pert; Mus musculus; RNA-Seq
Bone marrow derived lineage positive (B220+, CD3+, CD4+, CD8+, CD11b+, CD11c+,CD19+, Gr1+, NK1.1+, TER119+) cells from perturbed or unperturbed miR‑221/222 proficient and deficient mice were first depleted on magnetic column (LS from Miltenyi) using 4 mice in each condition. The BM single cell suspensions were labeled separately for preparative FACS, followed by staining with TotalSeq™-C anti-mouse antibodies: Hashtag‑1 for miR‑221/222 proficient and perturbed, Hashtag‑4 for miR‑221/222 proficient and unperturbed, Hashtag-2 for miR‑221/222 deficient and perturbed and Hashtag-5 miR‑221/222 deficient and unperturbed (BioLegned, #1: 155861, #2: 155863, #4: 155867, #5 155869). The unperturbed or perturbed miR221/222-proficient samples were pooled with deficient samples for scRNAseq. HSC was sorted as live, lineage(CD3, CD4, CD8, CD11b, CD11c, CD19, B220, Ly6g(Gr1), NK1.1, TER119)- CD117(ckit)+ Ly6a/e(Sca1)+ CD34- Flk2- CD150+ CD48- cells; MPP1 was sorted as live, lineage(CD3, CD4, CD8, CD11b, CD11c, CD19, B220, Ly6g(Gr1), NK1.1, TER119)- CD117(ckit)+ Ly6a/e(Sca1)+ CD34+ Flk2- CD150+ CD48- cells; MPP2 was sorted as live, lineage(CD3, CD4, CD8, CD11b, CD11c, CD19, B220, Ly6g(Gr1), NK1.1, TER119)- CD117(ckit)+ Ly6a/e(Sca1)+ CD34+ Flk2- CD150+ CD48+ cells. The samples were then applied to 10x Genomics workflow for cell capturing and scRNA gene expression (GEX) library preparation using the Chromium Single Cell 5′ Library & Gel Bead Kit as well as the Single Cell 5′ Feature Barcode Library Kit (10x Genomics). After cDNA amplification the CiteSeq libraries were prepared separately using the Single Index Kit N Set A while final GEX libraries were obtained after fragmentation, adapter ligation, and final Index PCR using the Single Index Kit T Set A. Chromium Single Cell 5′ Library & Gel Bead Kit as well as the Single Cell 5′ Feature Barcode Library Kit (10x Genomics).