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| Status |
Public on May 01, 2019 |
| Title |
ATACMe_THP1_2hrPMA_rep2 |
| Sample type |
SRA |
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| Source name |
THP-1 Monocyte
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| Organism |
Homo sapiens |
| Characteristics |
pma stimulation time point: 2 hr
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| Treatment protocol |
Differentiation of THP1 monocytes was induced with RPMI medium carrying PMA at a concentration of 100ng/mL (162nM). THP1 monocyte PMA stimulation time points were harvested with a combination of aspiration (for suspended cells) and trypsin (for adherent cells). The final collection of each time point was pelleted at 4°C, 500 R.C.F for 5 minutes. After pelleting cells were resuspended in ice cold PBS and cell counted using an automated cell counter (Biorad). This cell suspension was divided as input material for RNA extraction, genomic DNA extraction and ATAC reactions.
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| Growth protocol |
THP1 monocytes were cultured in RPMI medium (Gibco) supplemented with 10% fetal bovine serum, 2mM L-glutamine, 100 units penicillin/streptomycin and 1mM sodium pyruvate. During routine culture THP1 cells were maintained at a density of 0.5-2E6 cells/mL. All cells were regularly screened for mycoplasma contamination using the Lonza MycoAlert kit.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
THP1 cell suspension volume corresponding to 2E5 cells was pelleted in a 1.5mL tube at 4°C, 500 R.C.F for 5 minutes. Supernatant was aspirated and the pellet resuspended in 150µL cold ATAC lysis buffer (10mM Tris-HCl pH = 7.4, 10mM NaCl, 3mM MgCl2, 0.1% IGEPAL-630). This suspension was agitated by pipetting up/down with a 200µL micropipette tip 8 times. Subsequently, this suspension was pelleted at 4°C, 500 R.C.F for 10 minutes. Supernatant was discarded and pellet immediately resuspended in 190µL transposition reaction mix (10mM Tris-HCl pH=7.5, 5mM MgCl2, 10% Dimethylformamide) by pipetting up/down with a 200µL micropipette tip 3 times. After resuspension 10µL of Tn5 transposase pre-assembled with methylated adapters was added. Tube was flicked gently to mix all components and incubated at 37°C, 30 minutes, 700RPM in an Eppendorf Thermomixer. Immediately after the 30-minute incubation ATAC reactions were terminated by adding 1mL Zymo DNA binding buffer and vortexing. This binding buffer and ATAC reaction mixture was purified according to manufacturer instruction in the Zymo DNA Clean and Concentrator-5 kit. Final elution was in 12.5µL nuclease free H2O. After elution gap repair reactions were assembled and incubated as described in “Wang, Q. et al. Tagmentation-based whole-genome bisulfite sequencing. Nature Protocols 8, 2022-2032 (2013)“ with slight modification (11µL ATAC-Me DNA eluate, 2µL 10µM Tn5mC-Repl01 oligo, 2µL 10x ampligase buffer, 2µL dNTPs 2.5mM each)17. 2µL of the gap repair reaction was reserved for a test PCR amplification to confirm successful ATAC nucleosomal laddering. Subsequently, 2µL of 250mM EDTA (pH=8.0) was added to stop the reaction. Gap repaired ATAC-Me material was bisulfite converted according to manufacturer instructions using the EZ DNA Methylation-Lightning Kit (Zymo). Final elution was in 22µL of M-elution buffer supplied by the kit. Gap repaired, bisulfite converted ATAC-Me DNA reactions were PCR amplified with 12 cycles of amplification using HiFi HotStart Uracil + Ready Mix (KAPA) and barcoded primers. Post-amplification PCR reactions were cleaned and concentrated with a DNA Clean and Concentrator-5 column kit (Zymo). Elution was in 22µL nuclease free H2O. Preliminary library analysis for concentration and size distribution was performed using an Agilent 2200 TapeStation with a D5000 screentape.
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 4000 |
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| Description |
ATAC-Me-seq of THP1 Monocytes: 2hrs 162nM PMA treatment
ATAC-seq coupled with bisulfite conversion
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| Data processing |
Trimming: All sequencing library reads were trimmed of adapters using TrimGalore script wrapper for Cutadapt (Martin, 2011) and FastQC. Version: trim_galore 0.4.0. Command: trim_galore --fastqc --fastqc_args "--outdir ${FASTQC_DIR}" --clip_R1 9 --clip_R2 9 --paired --retain_unpaired --output_dir ${TRIM_DIR} ${R1} ${R2}
Mapping: Standard ATAC reads were mapped with bowtie2. Version: bowtie2 2.2.6. Command: bowtie2 -p 8 --no-discordant --no-mixed -X 1000 -x ${INDEX} -1 ${R1} -2 ${R2} -S ${MAPPED_DIR}/${MAPPED}.sam
Mapping: All bisulfite converted samples were mapped with WALT. Version: WALT v1.0. Command: walt -m 8 -t 6 -i ${INDEX} -1 ${R1} -2 ${R2} -o ${MAPPED_DIR}/${MAPPED}.sam
Mapping: All RNA-seq reads were mapped with STAR. Version: STAR_2.6.1a. Command: STAR --runThreadN 4 --genomeDir /data/hodges_lab/human_genome/GRCh38_Gencode24 --sjdbGTFfile /data/hodges_lab/human_genome/GRCh38_Gencode24/gencode.v24.primary_assembly.annotation.gtf --readFilesCommand zcat --readFilesIn --outSAMattrIHstart 0 --outSAMstrandField intronMotif --outFilterIntronMotifs RemoveNoncanonical --outSAMtype BAM SortedByCoordinate
ATAC peak calling: All peak calling was done with MACS2. Version: MACS2 2.1.1. Command: macs2 callpeak -t ${MAPPED_DIR}/${MAPPED}.shifted.bam -f BAMPE -n ${MACS_DIR}/${MAPPED} -g hs -q 0.005 -B --SPMR --keep-dup all --broad --broad-cutoff 0.005
DNA methylation analysis: The Methpipe suite of tools was used for all CpG methylation analysis. Version: Methpipe 3.4.3
Differential RNA-seq analysis: DESeq2 was use to calculate differential RNA expression across the time course. Version: DESeq2 1.18.1. R-Version: 3.4.3
Differential ATAC-seq analysis: TCseq was used to calculate differential chromatin accessibility across the time course. Version: 1.6.1. R-version: 3.4.3
Genome_build: hg19
Supplementary_files_format_and_content: Peak files are broadPeak output bed files from MACS2, meth files are a tab delimited text file of CpG methylation values for all symmetic CpGs in hg19, meth files are generated as output from Methpipe
Supplementary_files_format_and_content: DEseq2 normalized transcript abundance table
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| Submission date |
Apr 19, 2019 |
| Last update date |
May 02, 2019 |
| Contact name |
Emily Hodges |
| E-mail(s) |
emily.hodges@vanderbilt.edu
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| Organization name |
Vanderbilt University
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| Department |
Biochemistry
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| Lab |
Hodges
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| Street address |
2212 Garland Ave.
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| City |
Nashville |
| State/province |
TN |
| ZIP/Postal code |
37232 |
| Country |
USA |
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| Platform ID |
GPL20301 |
| Series (1) |
| GSE130096 |
ATAC-Me captures spatiotemporal dynamics of DNA methylation across the chromatin accessible genome |
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| Relations |
| BioSample |
SAMN11468131 |
| SRA |
SRX5713578 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM3731839_R1_ATAC-Me_THP1_PMA_Rep2A_120.meth.txt.gz |
126.6 Mb |
(ftp)(http) |
TXT |
| GSM3731839_R1_ATAC-Me_THP1_PMA_Rep2A_120_peaks.broadPeak.gz |
9.6 Mb |
(ftp)(http) |
BROADPEAK |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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