Illumina HiSeq 4000 sequencing; Single cell RNA-sequencing of lineage-negative fraction of stromal cells isolated from inguinal white adipose tissue in mouse
Single cell RNA-sequencing of lineage-negative fraction of stromal cells isolated from inguinal white adipose tissue in mouse
Tissue of male C57BL6J mice was harvested, digested, and stromal cells isolated via differential centrifugation. The lineage-negative fraction of stromal cells was purified via affinity column. Following centrifugation and removal of the medium, cells were resuspended at a concentration of 150–500 cells/μL. This cell suspension was mixed with C1 Cell Suspension Reagent (Fluidigm, Cat # 634833) at the recommended ratio of 3:2 immediately before loading 5 μL of this final mix on the C1 IFC. Cells were lysed via Module 2 mRNA-seq kit per Fluidigm protocol specifications (PN 100-7168). Resultant mRNA was stabilized and reverse-transcribed using the SMARTer kit (Clontech), also per Fluidigm protocol specifications. Approximate run time was ~7.75 hours (6.5 hours for lysis, reverse transcription, and amplification; and 1.25 hours for harvest). 3µL amplicons were diluted with 10µL C1 DNA Dilution Reagent (Fluidigm) to generate 13 µL mix, and transferred to a new plate. Sample concentrations were normalized manually, then prepared for successive tagmentation, amplification, and pooling per the Illumina Nextera XT DNA Library Preparation protocol guidelines.
Experimental Factor: organism part
inguinal adipose tissue
Experimental Factor: stimulus
none
Experimental Factor: single cell identifier
ING_(no-Tx)_single-21