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Sample GSM6124080 Query DataSets for GSM6124080
Status Public on Jun 06, 2022
Title BW10s5_PANC1_R1_5-Scr-0-4h1
Sample type SRA
 
Source name PANC1 cell line
Organism Homo sapiens
Characteristics cell line: PANC1
timepoint: 4h
condition: 0 TNF
knockdown: scr
Treatment protocol PANC-1 cells were transfected either with Ctrl or SIK3 specific siRNA and treated with 100 ng/ml rHuTNF for 4 h or 24h or left untreated.
Growth protocol Human pancreatic ductal adenocarcinoma cell line PANC-1 (ATCC® CRL-1469™; source: male). were acquired from American Type Cell Culture (ATCC). PANC-1 were cultured under standard conditions in DMEM media supplemented with 10% fetal calf serum, 100 U/ml penicillin G and 100 µg/ml streptomycin at 37 °C in a humidified atmosphere under 5% CO2.
Extracted molecule genomic DNA
Extraction protocol Chromatin accessibility mapping on purified cell populations was performed using the ATAC-seq method as previously described (Buenrostro et al., 2013; Corces et al., 2017), with minor adaptations. Briefly, in each experiment 2,000 - 15,000 sorted cells were pelleted by centrifuging for 10 min at 4 °C at 500 x g. After centrifugation, the pellet was carefully lysed in 50 µl resuspension buffer supplemented with NP-40 (Sigma), Tween-20 and Digitonin (10 mM Tris-HCl pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.1 % NP-40, 0.1 % Tween-20, 0.01 % Digitonin) and incubated for 3 minutes on ice. Then, 1 ml of ice-cold resuspension buffer supplemented with Tween-20 was added, and the sample was centrifuged at 4 °C at 500 x g for 10 minutes. The supernatant was discarded, and the cell pellet was carefully resuspended in the transposition reaction (25 µl 2 x TD buffer (Illumina), 2.5 µl TDE1 (Illumina), 16.5 µl PBS, 5 µl nuclease-free water, 0.5 µl 1% Digitonin (Promega), 0.5 µl 10% Tween-20 (Sigma)) for 30 min at 37 °C on a shaker at 1000 rpm. Following DNA purification with the Clean and Concentrator-5 kit (Zymo) eluting in 23 µl, 2 µl of the eluted DNA was used in a quantitative 10 µL PCR reaction (1.25 µM forward and reverse custom Nextera primers (Corces et al., 2017), 1x SYBR green final concentration) to estimate the optimum number of amplification cycles with the following program 72°C 5 min; 98°C 30 s; 25 cycles: 98°C 10 s, 63°C 30 s, 72°C 1 min; the final amplification of the library was carried out using the same PCR program and the number of cycles according to the Cq value of the qPCR. Library amplification using custom Nextera primers was followed by SPRI size selection with AmpureXP beads to exclude fragments larger than 1,200 bp. DNA concentration was measured with a Qubit fluorometer (Life Technologies). The libraries were sequenced using the Illumina HiSeq3000/4000 or NextSeq platforms.
 
Library strategy ATAC-seq
Library source genomic
Library selection other
Instrument model NextSeq 550
 
Data processing Sequenced reads were trimmed for adaptor sequences using skewer v0.2.2
Reads were mapped to hg38 whole genome using bowtie2 v2.3.3 with the –very-sensitive parameter and a maximum insert size of 2000 bp
Duplicate and unpaired reads were removed using the sambamba (Tarasov et al., 2015) ‘markdup’ command, and reads with mapping quality >30 and alignment to the nuclear genome were kept.
Peak calling for each sample was performed using HOMER v4.10 (Heinz et al., 2010) with the following approach: Two different peak sets were called, once we used the options ‘-style factor -fragmentLength 150 -size 250 -minDist 250 –L 4 –fdr 0.0001’ parameters, to identify highly robust small regions of open chromatin, and in a second approach we used the parameters ‘-region -fragmentLength 150 -size 150 -minDist 250 -L 4 -fdr 0.0001’ to identify larger regions of open chromatin. The two peak sets were then merged using the mergePeaks function of HOMER to create the peak set that was used for downstream analysis.
For visualization purposes only, coverage files from filtered bam files were produced using bedtools (Quinlan and Hall, 2010) genomeCoverageBed, each position was normalized by dividing to the total library size and multiplying by 106, followed by conversion to a bigwig using the bedGraphToBigWig command from the UCSC genome browser tools.
Assembly: UCSC hg38
Supplementary files format and content: Processed data files contain bed files (peaks) and bigWig files (for genome browser visuaizations)
 
Submission date May 09, 2022
Last update date Jun 06, 2022
Contact name Claudia GEBHARD
E-mail(s) claudia.gebhard@gmail.com
Organization name UNIVERSITY HOSPITAL REGENSBURG
Street address FRANZ-JOSEF-STRAUSS ALLEE 11
City REGENSBURG
ZIP/Postal code 93042
Country Germany
 
Platform ID GPL21697
Series (2)
GSE202304 Salt-inducible kinase 3 protects tumor cells from cytotoxic T cell attack by promoting TNF-induced NF-κB activation
GSE202305 RNA-seq and ATAC-seq of tumor resistance to immune cell attack
Relations
BioSample SAMN28160997
SRA SRX15210839

Supplementary file Size Download File type/resource
GSM6124080_BW10s5_PANC1_R1_5-Scr-0-4h1.bigWig 383.8 Mb (ftp)(http) BIGWIG
GSM6124080_BW10s5_PANC1_R1_5-Scr-0-4h1.intersect.peaks.bed.gz 597.2 Kb (ftp)(http) BED
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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