title: GSM4555587: Klf4_KO_unsorted01; Mus musculus; RNA-Seq
library_construction_protocol: RNA-seq: Individual atherosclerotic plaques from the brachiocephalic artery were removed from underlying media with forceps and deposited into FACS buffer (1% BSA in PBS) plus 1 ug/mL Actinomycin D,(Gibco, #11805017). Samples were then minced and digested with 1mL Liberase (Roche, #355374) plus 1ug/mL ActinomycinD for 1 hour at 37 degrees, then resuspended in PBS plus 0.04% UltraPure non-Acetylated BSA (ThermoFisher #AM2616) and filtered through 30um filters on ice. Samples were then either counted and submitted directly for scRNAseq or sorted by flow cytometry. Samples for flow were stained with Sytox Blue viability dye and sorted with a BD FACSAria Fusion cell sorter with gating on live, single cells that were tdTomato+, GFP+, or eYFP+. Cells were collected into LoBind tubes containing 0.04% non-acetylated BSA. RNA-seq: 2,000 cells in each group were targeted in Chromium 10X genomics libraries, which after barcoding, were pooled and sequenced on the Illumina MiSeq, 150 cycle high-output. ChIP-seq: Segments of the aorta from the arch to the aortic root and up to the carotid bifurcation isolated from 18 week western diet fed  SMC-Oct4+/+, and SMC-Oct4Δ/Δ (n = 15 each group) mice were snap-frozen in liquid nitrogen and then processed for ChIP assays using the  Oct4-specific antibody and sequenced using Illumina TruSeq Chip Library Kit and sequenced on the HiSeq2000. Sequencing reads were aligned to the mouse genome release M21 (GRCm38.p6) using the BOWTIE alignment tool39. These aligned reads were then processed involving removal of duplicate reads and format conversions using the SAMtools40 suite and converted into bam/bai format and loaded in the Integrative Genomics Viewer for visualization. The reads were also converted to BED format and peaks were identified using MACS241 callpeaks function against the mouse genome with a qvalue < 0.05. ChIP-seq: Illumina TruSeq Chip Library Kit according to the manufacturer's protocol and sequenced on the HiSeq2000
attributes.tag: GEO Accession
attributes.value: GSM4555587