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| Status |
Public on Dec 17, 2019 |
| Title |
Cal27 ATAC-seq |
| Sample type |
SRA |
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| Source name |
Tongue
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| Organism |
Homo sapiens |
| Characteristics |
tumor type: HNSCC
cell line: Cal27
tagmentation enzyme: TDE1 Tagment DNA Enzyme, Illumina, #FC-121-1038
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| Growth protocol |
GBM cell lines U87, T98G, LN229, LNZ308, SF767 and head and neck cell lines HN12, Cal27 and Detroit562 were maintained in DMEM (Hyclone, #SH30022.01) supplemented with 10% fetal bovine serum (Atlanta Biologicals, #S12450) and 1% penicillin-streptomycin (Gibco, #15140-122) and grown as adherent cultures. GBM neurosphere cell lines GSC23 and TS576 were maintained in DMEM/F12 (Gibco, #11320-033) supplemented with B27 supplement (Gibco, #12504-044) and 1% penicillin-streptomycin and grown in suspension.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Approximately 50,000 nuclei were resuspended in 20µL ice-cold Tagmentation Buffer, and incubated with 1µL Tagmentation enzyme (Illumina, #FC-121-1030) at 37 °C for 30 min with shaking 500 rpm. The tagmentated DNA was purified using MinElute PCR purification kit (Qiagen, #28004).
libraries were amplified using NEBNext High-Fidelity 2X PCR Master Mix (NEB, #M0541) with primer extension at 72°C for 5 min, denaturation at 98°C for 30s, followed by 8 cycles of denaturation at 98°C for 10s, annealing at 63°C for 30s and extension at 72°C for 60s. Amplified libraries were then purified using MinElute PCR purification kit (Qiagen, #28004), and two size selection steps were performed using SPRIselect bead (Beckman Coulter, #B23317) at 0.55X and 1.5X bead-to-sample volume rations, respectively. Each library was then sequenced on an Illumina NextSeq500 or HiSeq4000 to a depth of >= 20 million usable reads pairs
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| Library strategy |
ATAC-seq |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 4000 |
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| Data processing |
Nextera adapters were trimmed from the raw fastq files by using cutadapt with parameters “-m 5 -e 0.10 -a CTGTCTCTTATA -A CTGTCTCTTATA” and then aligned to human reference genome hg38 using bowtie2 with parameters “-X2000 --mm --local”
Improper mapped, poorly mapped and unmatched reads were filtered from the resultant raw bam files using samtools view with parameters “-F 1804 -f 2 -q30”
Duplicates were marked by “picard MarkDuplicate” and removed by “samtools view”
Final bam files were generated after removing mitochondrial reads by “awk” command
Replicate Bam files were merged using “samtools merge” and converted to tagAlign format using bedtools bamtobed with parameters “-bedpe -mate1 -I” and “awk”
To account for the cutting offset of Tn5 transposase, mapping position was shifted using “awk”
Peaks were called from tagAlign files using MACS2 callpeak with parameters “-g hs -p 0.01 --nomodel --shift -75 --extsize 150 -B --SPMR --keep-dup all --call-summits”
Bedgraph files output from MACS2 were converted to BigWig files using bedGraphToBigWig
NarrowPeak files from individual ATAC-seq replicates and pooled peak files from MACS2 were sorted for high P-value peaks (>= 13 -log10 [p < 0.05]) with “awk”
high P-value pooled peaks were intersected with high P-value peaks from each replicate using bedtools intersect with the -u parameter
In order to exclude promoter proximal peaks the Gencode hg38 annotation was downloaded and TSS +/- 2kb were filtered out with “awk”. High p-value peaks were intersected with the Gencode TSS annotation with bedtools intersect with the -v parameter
Promoter distal high p-value ATAC peaks were intersected with H3K27Ac ChIP-seq peaks from each specific cell line to identify ATAC peaks located within regions of H3K27Ac using bedtools intersect with the -wa parameter.
Motifs were identified from these peaks using HOMER findMotifsGenome with the parameter “-size given”
Genome_build: hg38
Supplementary_files_format_and_content: BigWig (bw) files contain IGV-viewable read density data, visualized as peaks. Browser Extensible Data (BED) files contain the subset of significant (p < 0.05), TSS distal, H3K27Ac overlapping peaks.
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| Submission date |
Mar 13, 2019 |
| Last update date |
Dec 17, 2019 |
| Contact name |
Nathan Jameson |
| E-mail(s) |
njameson@ucsd.edu
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| Organization name |
UC San Diego
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| Department |
Pathology
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| Lab |
Furnari
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| Street address |
9500 Gilman Dr
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| City |
La Jolla |
| State/province |
CALIFORNIA |
| ZIP/Postal code |
92093 |
| Country |
USA |
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| Platform ID |
GPL20301 |
| Series (2) |
| GSE128271 |
Intron 1-Mediated Regulation Of EGFR Expression In EGFR-Dependent Malignancies [ATAC-Seq] |
| GSE128275 |
Intron 1-Mediated Regulation Of EGFR Expression In EGFR-Dependent Malignancies |
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| Relations |
| BioSample |
SAMN11122076 |
| SRA |
SRX5517033 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM3670006_Cal27_ATAC.highPValue.distal_intersect.H3K27Ac.bed.gz |
480.2 Kb |
(ftp)(http) |
BED |
| GSM3670006_Cal27_Peaks_Pooled_treat_pileup.bw |
716.8 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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