title: GSM8453482: 22wk_human_fetal_outer_ear2_RNA; Homo sapiens; RNA-Seq
library_construction_protocol: Human fetal outer ear replicate 2 and epiglottis tissue were dissected from a second 22-week gestation fetus. Using scissors and a scalpel, the tissue was finely minced and dissociated by mechanical and enzymatic digestion: the tissue was incubated for 4 h with gentle agitation at 37oC in conical flasks containing 1 mg/mL collagenase II (Worthington), 1 mg/mL dispase (Gibco), 1:1000 gentamicin (Teknova), 1:500 primocin (Invivogen), 1% penicillin/streptomycin/amphotericin B (P/S/A; Corning), and 10% fetal bovine serum (FBS; Corning) in DMEM/F12 (Corning). The mixture was pipetted in the sterile hood every 30 min. The resulting cell suspension was passed through a 70-micron filter (Fisher) and washed in 2% FBS/PBS solution to stop enzymatic digestion. For red blood cell lysis, the cells were incubated in a solution consisting of 1 mL 2% FBS/PBS and 9 mL ammonium chloride solution (STEMCELL Technologies; cat. no. 07800) for 10 minutes on ice and washed in 2% FBS/PBS. We then resuspended the remaining cells in 10% dimethyl sulfoxide (DMSO) and 20% FBS in DMEM/F12 medium and cryopreserved at -80oC for one month. For human fetal outer ear replicate 1 and nose tissues, we additionally performed negative selection using the magnetic activated cell sorting system (Miltenyi Biotec) using a primary anti-CD45 monoclonal antibody, PE (eBioscience, 12-9459-42), and an anti-PE secondary antibody (Miltenyi, 130-048-801) to remove CD45+ white blood cells as per manufacturer's instructions, just prior to cryopreservation. Cells were thawed within a month of preservation for fluorescence activated cell sorting (BD SORP FACSAria IIu) to isolate live cells that exclude nuclear stain DAPI (Southern Biotech; cat. no. 0100-20) into 0.04% bovine serum albumin (BSA) in phosphate buffered saline (PBS) at 4oC. FACS-sorted cells were pelleted for 15 min (300 g at 4oC) and incubated in lysis buffer for 6 min on ice. Nuclei were isolated per manufacturer's instructions (10X Genomics, protocol CG000038, low cell input protocol), washed in Wash buffer and resuspended in Nuclei buffer. Nuclei count was confirmed using an automated cell counter (Countess II Cell Counter; Life Technologies) and nuclear integrity checked with a fluorescence confocal microscope with DAPI staining before use in library construction. We generated separate barcoded single-nuclei libraries with simultaneous profiling of gene expression and chromatin accessibility using 10X Genomics Chromium Single Nuclei Multiome Reagent Kit per manufacturer's instructions. snRNAseq + snATACseq