GSM7864375: Uninjured bio replicate 1 scATAC-seq; Mus musculus; ATAC-seq
The dorsal olfactory epithelium (OE) was surgically removed and dissociated: OE from each animal was individually processed in approximately 1 mL of pre-warmed (37°C) dissociation medium (150 units papain dissolved in 5 mL Neurobasal medium with 2.5 mM Cysteine and 2.5 mM ethylenediaminetetraacetic acid) with 100 units DNAse I and incubated at 37°C for 25 mins. Samples were then washed three times with 10% fetal bovine serum in phosphate buffered saline (PBS-FBS) and strained through a 35 μm nylon mesh filter cap into a 5 mL polypropylene tube to remove debris. Propidium iodide (PI) was added to cells at a final concentration of 2 ug/mL just before loading them onto a BD FACSAria cell sorter. After running negative controls (no YFP, no PI and no YFP, PI only), YFP-positive/PI-negative cells were collected in a low-binding 1.5 mL tube containing 10% FBS in PBS on ice. Cells were centrifuged at 500g for 5 min at 4°C. After supernatant removal, cells were resuspended in 200 μl 0.04% BSA in PBS and centrifuged again at 500g for 5 min at 4°C. 195 μl supernatant was removed, and 45 μl cold lysis buffer (10mM Tris-HCl pH 7.4, 10mM NaCl, 3 mM MgCl2, 0.1% Tween-20, 0.1% NP40, 0.01% Digitonin, 1% BSA) was added and mixed with the pellet by gently pipetting up and down 3 times. Samples were incubated on ice for 3 min, and 50 μl cold wash buffer (10mM Tris-HCl pH 7.4, 10mM NaCl, 3mM MgCl2, 0.1% Tween-20, 1% BSA) was added to each without mixing. Samples were centrifuged at 500g for 5 min at 4°C. 95 ul supernatant was carefully removed, 45ul cold 1X Nuclei Buffer was added to each sample without mixing, and samples were centrifuged again at 500g for 5 min at 4°C. 48 μl supernatant was removed without disturbing the pellet, which was then resuspended in the remaining (~5 μl ) buffer for use in single cell ATACseq. A Chromium Single Cell ATAC Library & Gel Bead Kit (PN-1000111) was used according to the manufacturer's instructions (10X Chromium Single Cell ATAC Reagents Kits User Guide Rev B). Briefly, 5 μl of each sample resuspended in 1X Nuclei Buffer was added to 10 μl Transposition Mix, mixed by pipetting 6 times, and incubated in a thermal cycler at 37°C for 1 hour. After addition of barcoding master mix, the Chromium Chip E Single Cell Kit (PN-1000086) and Chromium i7 Multiplex Kit N, Set A (PN-1000084) were used for GEM generation. Libraries were constructed using 13 rounds of PCR.