|
| Status |
Public on Feb 07, 2024 |
| Title |
Mo THP-1_5hmC [5hmC_Mo_3] |
| Sample type |
SRA |
| |
|
| Source name |
THP-1 Mo
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: THP-1
cell type: Monocytic cell line
antibody: anti-5hmC antibody (Diagenode)
|
| Treatment protocol |
Fragmentation efficiency was checked by running a Nano RNA Bioanalyzer chip (Agilent) on the Agilent 2100 Bioanalyzer. RNA was then incubated at 4C overnight with or without 12.5 μg of anti-5hmC antibody (Diagenode) in freshly prepared 1X Immunoprecipitation (IP) buffer (50 mM Tris-HCl pH 7.4, 750 mM NaCl and 0.5% Igepal CA-630, RNasin 400 U/ml and RVC 2 mM) supplemented with protease inhibitor (complete EDTA free, Roche). 60 μL of equilibrated Dynabeads Protein G (Invitrogen) were added to each sample and incubated for 2.5 hours at 4C. After three washes with 1X IP buffer, samples were eluted by addition of 1 mL of TriPure Reagent (Roche) as per manufacturer’s instructions.
|
| Growth protocol |
1 mg of DNAse treated total RNA was chemically fragmented by incubating RNA in fragmentation buffer (10 mM Tris-HCl pH7, 100 mM ZnCl2) at 94°C for 40 seconds, the reaction was stopped by the addition of 50 mM EDTA. Fragmented RNA was ethanol precipitated and resuspended in nuclease-free water.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was then incubated at 4C overnight with or without 12.5 μg of anti-5hmC antibody (Diagenode) in freshly prepared 1X Immunoprecipitation (IP) buffer (50 mM Tris-HCl pH 7.4, 750 mM NaCl and 0.5% Igepal CA-630, RNasin 400 U/ml and RVC 2 mM) supplemented with protease inhibitor (complete EDTA free, Roche). 60 μL of equilibrated Dynabeads Protein G (Invitrogen) were added to each sample and incubated for 2.5 hours at 4C. After three washes with 1X IP buffer, samples were eluted by addition of 1 mL of TriPure Reagent (Roche) as per manufacturer’s instructions.
6 pM of DNA library spiked with 1% PhiX viral DNA was clustered on cBot (Illumina) and then sequenced on a HiScanSQ module (Illumina).
|
| |
|
| Library strategy |
OTHER |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina HiScanSQ |
| |
|
| Data processing |
Rraw reads were trimmed to remove adaptor sequences and low-quality reads using Trimmomatic with the default setting. Read quality was assessed using FastQC. Clean reads were aligned to the human reference genome hg38 (ENSEMBL version 86) using the STAR aligner.
To minimise the rate of false positives, only uniquely mapped reads were selected using samtools.
Peaks enriched in immunoprecipitated over corresponding input samples were called as expected peaks using MACS2. Peaks enriched in input samples were called as observed (background) peaks using MACS2.
Peaks identified in both biological replicates were merged using the mergePeaks command in the HOMER software and overlapping peaks were mapped to the RefSeq gene annotation using intersectBed from BEDTools.
Enriched 5hmC motifs were identified using de novo motif search with the HOMER software (version 4.9.1). Motifs with the most significant P-values were visualised using WebLogo. DESeq2 (108) was used to identify 5hmC peak levels that were significantly different (1.5-fold) between two samples with a Benjamini-Hochberg correction (p <0.05).
bigWig files were generated using the deepTools bamCoverage by --normalizeUsing BPM and binsize=1. narrowPeak files were generated using MACS v2 with the parameters of -q 0.05.
Assembly: hg38
Supplementary files format and content: bigWig, narrowPeak
Library strategy: 5hmC-IP-Seq
|
| |
|
| Submission date |
Sep 12, 2022 |
| Last update date |
Feb 07, 2024 |
| Contact name |
RENHUA SONG |
| E-mail(s) |
Renhua.song1989@gmail.com
|
| Phone |
61452667663
|
| Organization name |
Centenary Institute
|
| Lab |
Epigenetics and RNA Biology Program
|
| Street address |
Charles Perkins Centre - D17, Level 4 West. The University of Sydney Johns Hopkins Drive
|
| City |
Camperdown |
| State/province |
NSW |
| ZIP/Postal code |
2050 |
| Country |
Australia |
| |
|
| Platform ID |
GPL15456 |
| Series (2) |
| GSE213203 |
5hmC modification of monocytes and macrophages |
| GSE213207 |
5hmC and m6a modification and Translatomes of monocytes and macrophages |
|
| Relations |
| BioSample |
SAMN30812476 |
| SRA |
SRX17536847 |
