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| Status |
Public on Aug 10, 2023 |
| Title |
atac_C57_Kupffer_LPS_2hr_rep2 |
| Sample type |
SRA |
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| Source name |
Kupffer Cells, C57BL6/J, LPS 2 hours
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| Organism |
Mus musculus |
| Characteristics |
source: Sorted non-parenchymal cells
cell type: Kupffer cell
strain: C57BL/6J
treatment: LPS 2 hours
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| Treatment protocol |
Male mice were fasted overnight and treated for 2 hours by intraperitoneal injection of 0.1 mg/kg E. coli O114:B4 lipopolysaccharide. Control mice were equivalently fasted but uninjected.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
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 (Seidman et al., 2020; Sakai et al., 2019). 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.
Transposase reactions and sequencing libraries were generated as described previously (Buenrostro et al., 2013; Sakai et al., 2019; Seidman et al., 2020) using 25,000 to 50,000 FACS purified Kupffer cells. Tagmented DNA was cleaned up using Zymo ChIP Clean & Concentrate columns and PCR amplified for 14 cycles using barcoding primers. Libraries were size selected to 175-225 bp using gel excision and purified as described (Texari et al., 2021). For F1 samples, dual indexed libraries were pooled for a targeted depth of 100 million reads per sample.
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| Library strategy |
ATAC-seq |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina NextSeq 500 |
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| Description |
atac_C57_Kupffer_LPS_2hr_rep2.peaks
atac_C57_Kupffer_LPS_2hr.idr
atac_f0_raw.txt
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| Data processing |
FASTQ files: Sequencing data were assessed for quality using fastqc. ATAC-seq data was mapped using Bowtie2 (Langmead and Salzberg, 2012). ATAC-seq data were trimmed to 30 bp to remove sequencing adapters, which improved mapping efficiency. Strain specific genomes for BALB/cJ and A/J were generated from by replacing invariant positions of mm10 sequence with alleles reported in the Mouse Genome Project strain specific VCF files. mm10 was used as the C57BL/6J strain specific genome. Samples from parental strains of mice were mapped to the strain specific genome. Mapped reads were shifted to the chromosome coordinates of the mm10 genome build using MARGE.pl shift with -ind set to balbcj or aj for reads mapped to the BALB/cJ or A/J genome, respectively (Link et al., 2018b)
F1 Hybrid: For samples from CB6F1/J samples, reads were mapped to the mm10 and BALB/cJ genome builds. Then the BALB/cJ mapped reads were shifted to the mm10 build with MMARGE as above. Perfectly mapped reads spanning genetic mutations between BALB/cJ and mm10 were identified using the MMARGE.pl allele_specific_reads command with -ind set to BALB/cJ and a second time with -ind set to mm10 resulting in two SAM files for each biological sample: one SAM file containing reads perfectly mapped to the mm10 genome that spanned known DNA sequence differences relative to the BALB/cJ genome; and a second SAM file containing reads perfectly mapped to the BALB/cJ genome that spanning known DNA sequence differences relative to the reference mm10 genome.
IDR: Strain specific ATAC-seq SAM files were used to generate HOMER tag directories and allelic IDR peaks were identified using each biological replicate. Alterations in allelic signals from pooled IDR peaks were detected using DeSeq2 and required the following thresholds: minimum normalized average tag depth > 16; absolute log2 fold-change > 1; and adjusted p-value < 0.05.
Assembly: mm10
Supplementary files format and content: Tab delimited file of IDR peaks
Supplementary files format and content: Tab delimited file of normalized and raw tag counts over IDR peaks.
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| Submission date |
Oct 20, 2022 |
| Last update date |
Aug 10, 2023 |
| Contact name |
Hunter Bennett |
| E-mail(s) |
h1bennet@health.ucsd.edu
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| Phone |
3603031396
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| Organization name |
University of California, San Diego
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| Department |
Dept. of Cellular & Molecular Medicine
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| Lab |
Christopher K. Glass Laboratory
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| Street address |
9500 Gilman Drive, MC 0651
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| City |
La Jolla |
| State/province |
California |
| ZIP/Postal code |
92093 |
| Country |
USA |
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| Platform ID |
GPL19057 |
| Series (2) |
| GSE216162 |
Discrimination of cell-intrinsic and environment-dependent effects of natural genetic variation on Kupffer cell epigenomes and transcriptomes [ATAC-Seq] |
| GSE216164 |
Discrimination of cell-intrinsic and environment-dependent effects of natural genetic variation on Kupffer cell epigenomes and transcriptomes |
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| Relations |
| BioSample |
SAMN31384045 |
| SRA |
SRX17966034 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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