GSM8956706: E16.5 epidermis, spinous cells, biol rep1; Mus musculus; RNA-Seq
K10-rtTA;TRE-H2B-GFP;K14-RFP pregnant dams were all fed with doxycycline chow at E12.5, and then sacrificed at either E14.5 or E16.5. Embryos were checked under a dissecting microscope for GFP and RFP expression before use, and their tails were taken to confirm genotypes. E16.5 and E14.5 back skins were cut off and treated with 1.4 U/ml dispase II (Roche, 4942078001) in HBSS at room temperature for 1 hour, then peeled off the epidermis from the dermis. The epidermis of E14.5 back skin was not peeled off due to its low thickness, but was still treated with dispase II to maintain the same conditions as the E16.5 samples. Both E16.5 epidermis and E14.5 skin samples were digested in 1:1 trypsin (GIBCO, 25200-056) with versene (GIBCO, 15040-066) at 37°C and rotated for 20 minutes. Samples were then mixed 1:1 with FACS buffer (HBSS with 2.5% FBS and 10 μg/ml DNAaseI) and centrifuged at low speed. The cell pellet was diluted into FACS buffer with propidium iodide solution (Sigma, P4864, to exclude dead cells), and filtered using sterile Celltrics 30 μm filters (Sysmex, 04-004-2326). For E16.5 samples, we excluded granular cells by using  Ulex europaeus agglutinin I lectin (UEA1), which binds to the more superficial cells. For this, we used biotinylated UEA1 (Vector Laboratories, B-1065) and detected them with APC-streptavidin (BioLegend, 405207).  The E14.5 intermediate cells (K10-GFP+;K14-RFP-;APC-UEA1-), E16.5 spinous cells (K10-GFP+;K14-RFP-;APC-UEA1-), and basal cells at E14.5 and E16.5 (K10-GFP-;K14-RFP+) cells were FACS sorted separately. RNA was extracted using a QIAGEN RNAeasy Mini kit (QIAGEN, 74104) following the manufacturer's protocols, with DNA digested using RNase-Free DNase (QIAGEN, 79254). Biological replicates, three independent RNA samples from each cell population, were collected and sent for sequencing and analysis by Novogene. NEB library: Total RNA was extracted and mRNA was purified using poly-T oligo-attached magnetic beads. The mRNA was randomly fragmented using fragmentation buffer, and first strand cDNA synthesis was performed using random hexamer primers and M-MuLV Reverse Transcriptase. Second strand cDNA was synthesized using DNA Polymerase I and RNase H. The resulting double-stranded cDNA was purified using AMPure XP beads, and end-repair was performed to generate blunt ends. After adenylation of 3′ ends, NEBNext adaptors with a hairpin loop structure were ligated. Size selection was performed to enrich for fragments of ~150–200 bp. Libraries were amplified by PCR and purified using AMPure XP beads.