title: GSM6585736: neclei isolated from LPS-treated mouse tongue epithelium; Mus musculus; ATAC-seq
library_construction_protocol: To isolate the nuclei for scATAC-seq, the peeled tongue epithelia from PBS- and LPS-treated mice were prepared. Six mice per group were used. Epithelia from the circumvallate papillae were cut off and rinsed in calcium-free Tyrode's buffer (140 mM NaCl, 5 mM KCl, 10 mM HEPES, 1 mM MgCl2, 10 mM glucose, 10 mM Na-pyruvate, 2 mM EGTA, pH 7.4). Circumvallate epithelia were then transferred to a solution containing 0.25% trypsin-EDTA (Fisher Scientific) and incubated at 37˚C for 15 min. Cold Opti-MEM with 10% fetal bovine serum (FBS) (Thermo Fisher Scientific) was added to the mixture, and cells were further dissociated by gentle pipetting. Cells were centrifuged, rinsed with cold Opti-MEM with 10% FBS, passed through a 40 µm cell strainer, and rinsed once with wash buffer (10 mM Tris-HCl, pH 7.5, 10 mM NaCl, 3 mM MgCl2, 1% bovine serum albumin (BSA), and 0.1% Tween-20). Dissociated cells were then resuspended in 0.1x Lysis buffer (10 mM Tris-HCl, pH 7.5, 10 mM NaCl, 3 mM MgCl2, 0.9% BSA, 0.01% Tween-20, 0.01% NP40, 0.001% digitonin) and incubated on ice for 5 min. Cell lysis was monitored by staining with trypan blue solution (0.4%; Thermo Fisher Scientific). Nuclei were rinsed with wash buffer, centrifuged, resuspended in 1x nuclei buffer (10x Genomics), and counted. About 4000 nuclei per group were used for downstream ATAC-seq procedures. The chromatin transposition reaction, nuclei partitioning, barcoding, and library construction were carried out using 10x Genomics Chromium scATAC reagent kits, following the manufacturer's protocols. High-throughput sequencing was also performed at the NGSC using Illumina HiSeq 4000 instruments. Cell Ranger ATAC 1.2.0