GSM3790766: D14 Large Wound Center scATAC-Seq; Mus musculus; ATAC-seq
Large wounds from adult mice (n = 3) were dissected and enzymatically dissociated with dispase (StemCell Technologies) for 20 mins at 37 oC to remove the epidermis. Remaining dermis was dissociated using 0.2% collagenase for 2 hours at 37oC. Cell suspensions were diluted with cold Hank's Buffered Salt Solution (HBSS; Life Technologies), strained through 40 µm cell filters (Falcon) and centrifuged at 280 X g. Liberated single cells were re-suspended in bovine serum album in HBSS before FACS purification. For FACS purification, a stringent forward and side scatter gates were used to exclude debris, and cell doublets. Following FACS, single nucleus was isolated, washed, and counted prior to use with the Chromium Single Cell ATAC protocol (as described in CG000169, Demonstrated Protocol for Nuclei Isolation ATAC Sequencing Rev B document at: https://support.10xgenomics.com/single-cell-atac/sample-prep/doc/demonstrated-protocol-nuclei-isolation-for-single-cell-atac-sequencing). Libraries were prepared according to 10X Genomics Chromium Single Cell ATAC Library & Gel Bead Kit Chemistry. Briefly, single nuclei were first treated in bulk with Tn5 transposase to preferentially insert sequencing adaptors into accessible DNA regions. Transposed nuclei are then partitioned into GEMs in the 10x Chromium Controller. All the DNA fragments from the same nucleus share a common 10x barcode. The barcoded, accessible DNA fragments are subsequently pooled for downstream processing and library preparation. Next generation sequencing was performed using Illumina HiSeq 4000 PE with a targeted sequencing depth of >10,000 median fragments per cell. 77.4% of the fragments overlapped a targeted region and fraction of transposition events in peaks was 53.2%.