|
| Status |
Public on May 01, 2019 |
| Title |
RNA_THP1_1hrPMA_rep2 |
| Sample type |
SRA |
| |
|
| Source name |
THP-1 Monocyte
|
| Organism |
Homo sapiens |
| Characteristics |
pma stimulation time point: 1 hr
|
| 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.
|
| 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.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
THP1 cells were harvested from each PMA stimulation time point by pelleting approximately 1E6 cells at 4°C, 500 R.C..F for 5 minutes. After removal of supernatant, cell pellet was homogenized with Trizol by repeatedly pipetting up/down with a 1mL micropipette tip. RNA was purified according to the Triozol manufacturere protocol. Libraries were prepared using manufacturer protocols in the SMARTer® Stranded Total RNA Sample Prep Kit - HI Mammalian kit available from Takara Bio USA, Inc (catalog # 634873)
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
| |
|
| Description |
RNA-seq of THP1 Monocytes: 1hrs 162nM PMA treatment
THP1_PMAtimecourse_RNAseq_DESeq2_NormalizedCountsTable.txt
|
| 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
|
| |
|
| Submission date |
Apr 19, 2019 |
| Last update date |
May 01, 2019 |
| Contact name |
Emily Hodges |
| E-mail(s) |
emily.hodges@vanderbilt.edu
|
| Organization name |
Vanderbilt University
|
| Department |
Biochemistry
|
| Lab |
Hodges
|
| Street address |
2212 Garland Ave.
|
| City |
Nashville |
| State/province |
TN |
| ZIP/Postal code |
37232 |
| Country |
USA |
| |
|
| Platform ID |
GPL24676 |
| Series (1) |
| GSE130096 |
ATAC-Me captures spatiotemporal dynamics of DNA methylation across the chromatin accessible genome |
|
| Relations |
| BioSample |
SAMN11468113 |
| SRA |
SRX5713596 |
