 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on May 05, 2020 |
| Title |
ATAC_KC-N_rep1 |
| Sample type |
SRA |
| |
|
| Source name |
Sorted hepatic macrophages
|
| Organism |
Mus musculus |
| Characteristics |
strain: C57BL/6J
cell type: Sorted hepatic macrophages
cell subtype: F4/80-Hi Tim4-Pos Kupffer cells
treatment: NASH diet
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Hepatic macrophages: Mice were humanely euthanized by exposure to CO2 and liver non-parenchymal cells processed for fluorescence activated cell sorting of Kupffer cells, with modifications from published methodology (Mederacke et al., 2015; Muse et al., 2018; Seki et al., 2007). In brief, livers were retrograde perfused for 3 min at a rate of 5-7 ml/min through the inferior vena cava with HBSS without Ca++ or Mg++ supplemented with 0.5 mM EGTA, 0.5 mM EDTA, and 20 mM HEPES. Perfusions were then switched to 40 ml of a digestion buffer, held at 37C, comprised of HBSS with Ca++ and Mg++ supplemented with 0.033 mg/ml of Liberase TM (Roche), 20 μg/ml DNaseI (Worthington), and 20 mM HEPES. Livers were then excised, minced, and digested for an additional 20 minutes in vitro at 37C with gentle rotation in 20 ml of fresh digestion buffer. The perfusion and digestion steps were performed in the presence of 1 μM flavopiridol to offset transcriptional changes associated with digestion. After tissue digestion, cells were passed through a 70-micron cell strainer and hepatocytes removed by 2 low-speed centrifugation steps at 50 X G for 2 min. Nonparenchymal cells in the supernatant were further separated from debris by pelleting for 15 min at 600 X G in 50 ml of 20% isotonic Percoll (Sigma Aldrich) at room temperature. Cells were then washed from Percoll containing buffer and suspended in 10 ml 28% OptiPrep (Sigma Aldrich) and carefully underlaid beneath 3 ml of wash buffer. The resulting gradient was centrifuged at 1,400 X G for 25 minutes at 4C with no break and cells enriched at the interface were saved and subjected to isotonic erythrocyte lysis. Enriched non-parenchymal cells were then washed, suspended in PBS, then stained for 10 minutes with Zombie NIR (BioLegend) and purified anti-CD16/32 (93, BioLegend) to label dead cells and block Fc receptors. Cells were then immunolabeled with specific antibodies of interest, washed, and sorted using a Beckman Coulter MoFlo Astrios EQ configured with spatially separated 355 nm, 405 nm, 488 nm, 561 nm, and 642 nm lasers.
Peripheral monocytes: Mice were humanely euthanized by exposure to CO2. Blood was collected from mice via cardiac puncture into K3EDTA tubes and subjected to RBC lysis (eBioscience). Cells were pelleted by centrifugation at 350 X G for 10 minutes at 4°C and washed, suspended in PBS, then stained for 10 minutes with Zombie NIR (BioLegend) and purified anti-CD16/32 (93, BioLegend) to label dead cells and block Fc receptors. Next, cells were stained in wash buffer for an additional 20 minutes with the antibodies listed in the Key Resource Table. Stained cells were washed twice and strained through a 30 μm strainer, then subjected to cell sorting using a Beckman Coulter MoFlo Astrios EQ configured with 355 nm, 405 nm, 488 nm, 561 nm, and 642 nm lasers. Each cell population was hierarchically gated using Beckman Coulter Summit software. Ly6CHi monocytes were defined as CD45PosCD11bHiCD115PosCD19NegCD90.2NegLy6GNegLy6CHi. Ly6CHi monocytes were further restricted to single particles by comparing height and area side scatter pulses, and dead cells were excluded by detecting the integration of the live/dead dye (Zombie NIR).
Approximately 50,000 sorted cells were washed once with PBS and once with 10mM Tris pH 7.4, 10 mM NaCl, 3 mM MgCl2, and 0.1% Igepal by centrifugation at 500 X G at 4C. Cells were then suspended in 50 μl reaction buffer comprised of 25 μl Tagment DNA buffer, 2.5 μl Tagment DNA enzyme, and 22.5 μl nuclease free water, using reagents sourced from Illumina Nextera DNA Library Prep Kit (Buenrostro et al., 2013). Transposase reactions were carried out at 37C for 30 minutes and immediately DNA was purified using Zymo ChIP Clean & Concentrate columns. Resulting DNA was PCR amplified for 14 cycles using barcoding primers and resulting libraries were size selected by gel excision to 175-225 bp as described in (Link et al., 2018). Library DNA was purified, and single end sequenced using a HiSeq 4000 or a NextSeq 500.
|
| |
|
| Library strategy |
ATAC-seq |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina NextSeq 500 |
| |
|
| Description |
processed data file: KC-N_ATAC_IDR.txt
|
| Data processing |
FASTQ files from sequencing experiments were mapped to the mouse mm10 genome. Bowtie2 with default parameters was used to map ATAC-seq experiments (Langmead and Salzberg, 2012). HOMER was used to convert aligned reads into “tag directories” for further analysis (Heinz et al., 2010).
IDR analysis: ATAC-seq experiments were performed in replicate. Peaks were called with HOMER for each tag directory with parameters -L 0 -C 0 -fdr 0.9 -minDist 200 - size 200. IDR (Li et al., 2011) was used to test for reproducibility between replicates, and only peaks with IDR < 0.05 were used for downstream analysis. The pooled tag directory from two replicates was used for track visualization.
To quantify transcription factor chromatin accessibility, peak files were merged with HOMER’s mergePeaks and annotated with raw tag counts with HOMER’s annotatePeaks using parameters -noadj, -size given. To annotate H3K27ac signal around ATAC-seq peaks the parameter -size 2000 was used. Subsequently, DESeq2 (Love et al., 2014) was used to identify the differential H3K27ac signal or chromatin accessibility with > 2 fold-change and p-adj < 0.05, unless stated otherwise in the text.
To identify motifs enriched in peak regions over the background, HOMER’s motif analysis (findMotifsGenome.pl) including known default motifs and de novo motifs was used. The background sequences were either from random genome sequences or from peaks from the comparing condition indicated in the main text and in the figure legends.
Genome_build: mm10 (GRCm38)
Supplementary_files_format_and_content: *_IDR.txt: Tab-delimited text files include IDR peak files for each set of replicates (as described above) as well as peak files on each individual replicate with HOMER with parameters -minDist 200 - size 200.
|
| |
|
| Submission date |
May 03, 2020 |
| Last update date |
May 07, 2020 |
| Contact name |
Christopher K Glass |
| E-mail(s) |
ckg@ucsd.edu
|
| Organization name |
University of California, San Diego
|
| Department |
School of Medicine
|
| Street address |
9500 Gilman Drive
|
| City |
La Jolla |
| State/province |
CA |
| ZIP/Postal code |
92093 |
| Country |
USA |
| |
|
| Platform ID |
GPL19057 |
| Series (2) |
| GSE128335 |
Niche-Specific Re-Programming of Epigenetic Landscapes Drives Myeloid Cell Diversity in NASH [ATAC-seq] |
| GSE128338 |
Niche-Specific Re-Programming of Epigenetic Landscapes Drives Myeloid Cell Diversity in NASH |
|
| Relations |
| BioSample |
SAMN14823895 |
| SRA |
SRX8244459 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
|
|
|
|
 |
