title: GSM3676085: Niche_LepR-tdT; Mus musculus; RNA-Seq
library_construction_protocol: For sample 1-3, red cell-depleted WBM cells were first enriched using lineage depletion kit (Miltenyi Biotec, Germany) and then stained with antibodies against cKit PE-Cy7, Sca1 PE, CD34 Alexa 647 for 90 minutes on ice followed by streptavidin eF450 for 20 minutes on ice. 4',6-diamidino-2-phenylindole (DAPI) was used to check viability. For sample 3-4, the mice were injected intravenously with VE-Cadherin A647 (BioLegend, CA). WBM plug was obtained by flushing tibias and digestion with Collagenase I (Worthington Biochemical Corporation, NJ), Dispase II (Roche Applied Science, Swizerland) and DNase I (Sigma-Aldrich) in HBSS with calcium and magnesium. The digested marrow was pooled with staining buffer (HBSS +2% FBS +2mM EDTA) to stop the enzymatic reaction. The red cell-depleted WBM cells were stained with antibodies against CD45 eFluor 450, Ter119 eFluor 450, and biotinylated LepR (1:100, R&D Systems, CA) on ice for 30 minutes followed by streptavidin PE-Cy5 on ice for 15 minutes. Subsequent cDNA purification, amplification (12 cycles) and library construction (sample index PCR 14 cycles) was performed as instructed Sample libraries were sequenced on the Illumina NovaSeq 6000 system using S1 flow cell (Illumina)
attributes.tag: GEO Accession
attributes.value: GSM3676085