|
| Status |
Public on Apr 16, 2024 |
| Title |
ATAC_control_IL4_2h_R2 |
| Sample type |
SRA |
| |
|
| Source name |
mouse bone marrow derived macrophages
|
| Organism |
Mus musculus |
| Characteristics |
cell type: BMDM
treatment: 1h DMSO followed by 2h IL4 10ng/mL
genotype: wild type
|
| Treatment protocol |
LPS (10 ng/ml) treatment for 30min, 1h, 2h or 4h, IL4 (10 ng/ml) for 30min, 1h, 2h or 4h
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
genomic DNA
5 × 104 BMDM cells were pelleted by centrifugation and resuspended in 50 µl of lysis buffer (10 mM Tris-HCl (pH 7.4), 10 mM MgCl2, 0.1% Igepal CA-630), gently pipetting up and down 10 times. Nuclei were pelleted by centrifugation for 10 min at 500g at 4 °C and resuspended in 25 µl of reaction buffer containing 1 µl of Tn5 transposase made in-house using a described protocol (Picelli et al. 2014a), 5 µl of 5x transposase buffer (50 mM Tris-HCl, (pH 8.4) and 25 mM MgCl2), and 19 µl of milliQ water. The tagmentation mix was incubated at 37 °C for 60 min gently shaking, in order to avoid precipitation of nuclei. The reaction was stopped by the addition of 5 µl of cleanup buffer (900 mM NaCl, 30 mM EDTA), 2 µl of 5% SDS and 2 µl of Proteinase K (20 µg /µl) (Merck cod. P2308) and incubation for 30 min at 40 °C. Tagmented DNA was isolated using 2× Agencourt Ampure Xp beads (Beckman cod.A63881) and amplified by PCR with the HotStart Kapa HiFi enzyme (Roche cod. 07958889001). Fragments smaller than 600 bp were isolated by negative size selection (using 0.65× Agencourt Ampure Xp beads) and then purified with 1.8× Agencourt Ampure Xp beads. Libraries were single-end sequenced (76 bp) on an Illumina NextSeq500 platform.
|
| |
|
| Library strategy |
ATAC-seq |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina NextSeq 500 |
| |
|
| Description |
ATACseq
ATAC_reference_peaks.bed
ATAC_reference_peaks_extended.bed
ATAC_reference_summits.bed
|
| Data processing |
Reads were quality filtered according to the illumina pipeline
Following adapter trimming, single end reads were trimmed with trimmomatic (Bolger et al. 2014) (version 0.39 flags: EADING:3 TRAILING:3 SLIDINGWINDOW:4:15 MINLEN:38).
Resulting reads were mapped with ShortStack (Axtell 2013).
Resulting bam files were then filtered using SAMtools (version 1.9) in order to remove unmapped reads, failing quality or mapping to mitochondrial chromosome.
Reads were then shifted of 4nt on the positive strand and -5nt on the negative strand using basic awk operations (Buenrostro et al. 2013).
Peak calling was performed using MACS2 (version 2.2.6; options --nomodel --extsize 146 --nolambda --keep-dup 'all' --call-summits). A reference set of peaks was created by selecting peaks called in each samples/replicate with qvalue ≤ 0.01 and being consistent between replicates (overlapping area of at least 50% between replicates).
The resulting set of peaks was used to count reads in each sample using the R/Bioconductor package GenomicRanges and GenomicAlignment (Lawrence et al. 2013).
Sample normalization was achieved by selecting invariant ATAC peaks across samples (Gualdrini et al. 2016). Briefly, we modelled the frequency distribution of the differences in reads counts across samples with a log-normal distribution. Peaks laying within 1σ of the best fitted mean difference were considered as invariant and used to normalize the samples.
Assembly: mm10
Supplementary files format and content: .bw are BigWig normalised files
|
| |
|
| Submission date |
Nov 29, 2022 |
| Last update date |
Apr 16, 2024 |
| Contact name |
Francesco Gualdrini |
| E-mail(s) |
Francesco.gualdrini@ieo.it
|
| Organization name |
European Institute of Oncology
|
| Department |
Department of Experimental Oncology
|
| Lab |
Transcriptional Control in Inflammation and Cancer
|
| Street address |
Via Adamello, 16
|
| City |
Milan |
| ZIP/Postal code |
20139 |
| Country |
Italy |
| |
|
| Platform ID |
GPL19057 |
| Series (2) |
| GSE218966 |
Unbiased profiling of clinical kinase inhibitors’ effects in activated macrophages using chromatin modifications as high-content readouts [ATAC-Seq] |
| GSE219240 |
Unbiased profiling of clinical kinase inhibitors’ effects in activated macrophages using chromatin modifications as high-content readouts |
|
| Relations |
| BioSample |
SAMN31925454 |
| SRA |
SRX18411617 |
