 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on Jul 01, 2022 |
| Title |
MG_ATAC_MIA_LPS_Rep1 |
| Sample type |
SRA |
| |
|
| Source name |
brain striatum
|
| Organism |
Mus musculus |
| Characteristics |
tissue: brain striatum
cell type: microglia
group: MIA
treatment: LPS
Sex: male
number of_cells: 20069
ID: 19-25-3-MIA-LPS
|
| Treatment protocol |
Lipopolysaccharide (LPS) was delivered by intraperitoneal injection to mice at a dose of 4500 endotoxin units / gram body weight. Microglia were extracted 18 hours after LPS or saline treatment.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Male mice (8-10 wk) were deeply anesthetized with ether, perfused with PBS, and brains extracted. The meninges were removed and then the frontal cortex and striatum were collected into 2 mL tubes with HBSS on ice. The anterior striatum was collected from a coronal slice anterior to 1.7 mm bregma by collecting the tissue surrounding the nucleus accumbens. The next coronal slice was cut at -0.5 bregma and the dorsal and ventral striatum was excised from this slice, excluding the olfactory tubercle, and all striatum pieces were pooled together. The brain pieces were diced with scissors and then homogenized with papain according to the adult neural dissociation kit (Miltenyi). Briefly, the tissue was treated with papain and DNase at 37°C for 30 min with rotation. The homogenates were pipetted gently and filtered with a 70 µm strainer. The cell pellet was resuspended in debris removal solution (300 µL debris removal, 700 µL PBS), overlaid with 300 µL of PBS, centrifuged at 3000xg for 10 min, debris aspirated, washed with PBS, and centrifuged at 1000xg for 10 min. The cells were resuspended in 0.5% BSA in HBSS. For fluorescence-activated cell sorting the cells were stained with CD45-APC, CD11b-BV421, and P2RY12-PE antibodies for 30 min at 4C. The cells were washed with buffer and sorted for live (PI-), CD11b+, CD45-low, P2RY12+ before final collection into ATAC collection buffer.
The ATAC libraries were prepared according to Corces et al. 2017. Briefly, cells were pelleted by spinning at 500g for 15 min at 4C and the supernatant carefully removed. 50 ul of cell lysis buffer was added (10 mM Tris-HCl, 10mM NaCl, 3 mM MgCl2, 0.1% NP-40, 0.1% Tween-20, 0.01% Digitonin), pipetted to mix 3-5 times, incubated on ice for 3 min, and washed (10 mM Tris-HCl, 10mM NaCl, 3 mM MgCl2, 0.1% Tween-20). The nuclei were spun to pellet at 500 g for 30 min at 4C and the supernatant carefully removed. Then the Tn5 buffer (1X TD Buffer, 1x PBS, 0.1% Tween-20, 0.01 Digotinin, 1 ul Tn5) was added and incubated at 37°C for 1 hour with 1000 RPM shaking. The DNA was collected using the Qiagen Minelute cleanup kit. The DNA was eluted with 11 ul EB Buffer. For library preparation, 10 ul of prepared DNA was mixed with 1.25 uM indexing primers and 1x NEBNext High-Fidelity PCR Master Mix and amplified (72C 5 min, 98C 30 sec, 5 cycles: 98C 10 sec, 63C 30 sec, 72C 1 min). 5 ul was used for qPCR to test amplification and calculate additional cycles. 6-8 more cycles were run before cleanup with Ampure XP beads. The libraries with quality control checked using a Bioanalyzer then pooled at equal mass for sequencing on a Novaseq6000.
|
| |
|
| Library strategy |
ATAC-seq |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina NovaSeq 6000 |
| |
|
| Description |
S14
|
| Data processing |
Alignment: FASTQ files were trimmed with CUTADAPT ( cutadapt -a CTGTCTCTTATA -A CTGTCTCTTATA --cores=0 --pair-filter=any -m 20 ). Trimmed FASTQ files were aligned and assembled with bowtie2 ( bowtie2 -p 8 --very-sensitive-local -X 2000 -x ${index_path}/mm10 )
Cleanup: Mitochondrial and Duplicates reads were tagged and removed with picard (picard MarkDuplicates VALIDATION_STRINGENCY=LENIENT ASSUME_SORTED=true REMOVE_DUPLICATES=false ) and samtools (samtools view -h -q 30 -F 1804 -f 2 ${in_path}/mark.bam | awk '{if(!match($3, /chrM/)){print $0}}' | awk '{if(!match($3, /[_]/)){print $0}}' | samtools view -hb > ${out_path}/clean.bam)
Peaks: Peaks were called with MACS2 ( macs2 callpeak -t ${in_path}/clean.bam -f BAMPE --min-length 100 --max-gap 50 --bdg -g mm --keep-dup all -n SID --outdir ${out_path}/ --tempdir ${tmp_path}/ )
Differential accessibility was performed with DiffBind and permutation tests.
Assembly: mm10 (GRCm38)
Supplementary files format and content: narrowPeak files
|
| |
|
| Submission date |
Apr 28, 2022 |
| Last update date |
Jul 03, 2022 |
| Contact name |
Lindsay N Hayes |
| E-mail(s) |
lhayes14@jhmi.edu
|
| Organization name |
Johns Hopkins University
|
| Department |
Neuroscience
|
| Street address |
600 North Wolfe Street, Meyer 4-136
|
| City |
Baltimore |
| State/province |
Maryland |
| ZIP/Postal code |
21287 |
| Country |
USA |
| |
|
| Platform ID |
GPL24247 |
| Series (2) |
| GSE201815 |
Prenatal immune stress blunts microglia reactivity which impairs neurocircuitry [Adult_ATAC-seq] |
| GSE201817 |
Prenatal immune stress blunts microglia reactivity which impairs neurocircuitry |
|
| Relations |
| BioSample |
SAMN27959616 |
| SRA |
SRX15041520 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM6072090_S14_peaks.narrowPeak.gz |
1.2 Mb |
(ftp)(http) |
NARROWPEAK |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
|
|
|
|
 |
