GSM7835700: Bulk RNAseq of nasal OSN cells (STAT6). Treatment: Naive and sample id: 221014_G7; Mus musculus; RNA-Seq
Then, the nasal mucosa was separated from the bones mechanically and incubated in Tyrode's Solution with bicarbonate, HEPES, 0.25% BSA, without Ca2+ and added 31 U/ml of papain, 0.25 mg/ml L-cysteine and 20 μg/ml DNase I for 40 min at 37 °C on a rotating shaker at 220 rpm. The papain enzymatic activity was terminated with Tyrode's Solution with HEPES and Ca2+ and with 0.01 mg/ml leupeptin and the digested mucosa was triturated thoroughly and prepared for flow cytometric staining. All samples were pre-incubated with TruStain FcX™ (Biolegend) at 1µg per 106 cells in 100 µl volume for 10 minutes prior to immunostaining (Fig. S1A). 6,000-10,000 cells per sample were collected into PBS and 0.04% (w/v) bovine serum albumin (BSA) and submitted for scRNA-seq. Single-cell suspensions for low input (bulk) RNA-sequencing at 1,000 cells per sample were sorted into TCL buffer (Qiagen) supplemented with 1% 2-mercaptoethanol. We analyzed a total of 10 mice, treated with PBS (n=4), with one Alternaria challenge (n=4), and with two challenges (n=2). Sorted samples, containing 6,000-10,000 cells each, were submitted for droplet-based single-cell RNA-sequencing (10X Genomics) to the Single Cell Genomic Core at Brigham and Women's Hospital. Cells were washed and resuspended in PBS and 0.04% (w/v) bovine serum albumin (BSA) per the manufacturer's guidelines. Purified samples were run through 10X controller and individually bar-coded libraries were generated and pooled. Pooled library samples were sequenced on a NovaSeq S1 (Illumina) in partnership with Dana Farber Cancer Institute (DFCI) Molecular Biology Core Facilities. 10X Chromium V3 Chemistry Single-cell RNA Seq