GSM7415241: Ana_HF_Setdb1_Het; Mus musculus; RNA-Seq
Following euthanasia, back skin at anagen stage was removed and submerged in 0.25% collagenase/HBSS (Gibco) containing DNase I for 1hr at 37°C. Release cells were scraped off, resuspended in cold PBS, and the remaining skin was then incubated with 0.25% trypsin-EDTA (Gibco) for 10 min at 37C, neutralized by cold PBS containing 2% calcium-chelated fetal bovine serum FBS(-), and filtered through a 40-μm cell strainer. Cells were spin down at 400g for 10 min at 4°C, and washed once with cold PBS containing 2% FBS(-). Freshly isolated cells were incubated with DAPI and sorted using a 70-μm nozzle on a BD FACSAria equipped with FACSDiva software (BD Biosciences), gating on DAPI- to isolate live cells. Library was performed according to the manufacter's instructions (single cell 3' v3.1 protocol, 10x Genomics). Briefly, live cells seperated from mouse back skin were resuspended in the master mix and loaded together with partitioning oil and gel beads into the chip to generate the gel bead-in-emulsion (GEM). The poly-A RNA from the cell lysate contained in every single GEM was retrotranscripted to cDNA, which contains an Ilumina R1 primer sequence, Unique Molecular Identifier (UMI) and the 10x Barcode. The pooled barcoded cDNA was then cleaned up with Silane DynaBeads, amplified by PCR and the apropiated sized fragments were selected with SPRIselect reagent for subsequent library construction. During the library construction Ilumina R2 primer sequence, paired-end constructs with P5 and P7 sequences and a sample index were added.