GSM8759378: PADI4 cKO skin P1; Mus musculus; RNA-Seq
At postnatal day 1, K14-Cre Padi4 fl/fl mouse back skins were dissected and and put in 1:1 1xDispase:PBS (from 5x (10mg/ml) Dispase stock) at 4°C overnight with the dermal side facing the enzymatic solution. After incubation the epidermis and dermis were mechanically separated using fine forceps. The dermal fraction, containing the growing hair follicles, was incubated put in HBSS (Thermofisher Scientific (Gibco)) with 20% collagenase (in PBS (Sigma-Aldrich)) at 37°C for 1 h. After incubation, the dermal/hair follicle fraction was scraped off using a scalpel and collected by centrifugation at 300xg for 10 min, after which the pellet was resuspended in trypsin (Thermofisher Scientific (Gibco)) and incubated for 15 min at 37°C. Cell suspensions were then filtered using both 70- and 40-mm filters, washed twice in PBS and pelleted by centrifugation for 5min at 300xg. DAPI was used to exclude dead cells. Epithelial cells were tdTOMATO positive. Using a fluorescence activated cell sorter (BD FACS AriaII), approximately 100,000 sorted tdTOMATO-positive cells from each animal were collected in E-low medium without serum for subsequent single-cell library preparation. Single-cell libraries were prepared using the Chromium Next GEM Single Cell 3' v3.1 Reagent Kits (10x Genomics, PN-1000269) according to the manufacturer's protocol (CG000315 Rev E). Single-cell suspensions were adjusted to achieve a target recovery of approximately 10,000 cells. Cells were loaded onto a Chromium Next GEM Chip G along with barcoded gel beads, master mix reagents, and partitioning oil to create Gel Beads-in-Emulsion (GEMs) using the Chromium Controller. Each GEM contained a unique 10x barcode and unique molecular identifier (UMI) linked to mRNA transcripts through reverse transcription. GEMs were incubated to perform reverse transcription, after which they were broken, and cDNA was recovered using Dynabeads with Dynabead cleanup buffer (Ref no). First-strand cDNA was amplified by PCR to generate sufficient material for library construction. The amplified cDNA was subjected to enzymatic fragmentation, end-repair, and A-tailing to optimize fragment size. Adapters were ligated, and sample indices were introduced via sample index PCR. Cleanup steps were performed throughout the process to purify and size-select cDNA and libraries. Library quality and concentration were assessed using an Agilent Bioanalyzer with High Sensitivity DNA reagents. The final libraries consisted of paired-end constructs with dual indices. Libraries were pooled and sequenced to obtain paired-end reads, with Read 1 capturing the 10x barcode and UMI information and Read 2 covering the cDNA insert.