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| Status |
Public on Jan 22, 2024 |
| Title |
DNMT3B_KO_DNMT3B_W263A_exp_2_WGBS |
| Sample type |
SRA |
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| Source name |
HCT116 with knocked out DNMT3B expressing DNMT3B W263A
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| Organism |
Homo sapiens |
| Characteristics |
cell line_background: HCT116
cell type: colorectal cancer cell line
genotype: DNMT3B KO
treatment: Expressing DNMT3B W263A
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| Treatment protocol |
To express DNMT3B isoforms, HCT116 and DNMT3B KO cells were transfected with FuGENE HD transfection reagent (Promega). For stable integrants, DNMT3 expression constructs were co-transfected with a plasmid expressing piggyBac transposase. After 48 h cells stably expressing DNMT3s were selected with 1 μg/ml puromycin and expanded in the presence of puromycin for 3-4 weeks before being harvested for analysis. Knock-in mutations were introduced by CRISPR editing.
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| Growth protocol |
HCT116 and DNMT3B cells were gifts from B. Vogelstein (Rhee et al 2002 PMID: 11932749). Cells were cultured in McCoy’s 5A medium (Gibco) supplemented with 10% fetal calf serum (Life technologies) and penicillin-streptomycin antibiotics at 140 and 400 µg/ml respectively.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
To extract genomic DNA cells were resuspended in genomic lysis buffer (300 mM NaCl, 1% SDS, 20 mM EDTA) and incubated with Proteinase K (Roche) at 55 C overnight. RNA was removed by incubation with RNase A/T1 Cocktail (Ambion) at 37°C for 1 h, in between two phenol-chloroform extraction steps. DNA was quantified by Nanodrop 8000 spectrophotometer and purity assessed by electrophoresis.
100 or 200 ng of purified DNA samples were sheared using the Covaris E220 Evolution Focused Ultrasonicator to create fragments of approximately 350 bp. 0.5 ng of unmethylated phage-λ DNA (NEB) was spiked into each DNA sample prior to shearing to allow assessment of the efficiency of the bisulfite-conversion reaction. 100 ng of each sheared DNA sample was then processed using the EZ DNA Methylation Gold Kit or EZ DNA Methylation-Lightning Kit (Zymo Research) according to the manufacturer’s protocol to create bisulfite-converted single-stranded (BC-ss) DNA. For WGBS experiment 1, the TruSeq DNA Methylation Kit (Illumina Inc.) was used. Bisfulfite converted single-stranded DNA was randomly primed with a polymerase able to read uracil nucleotides to synthesise DNA strands containing a sequence specific tag. 3’ ends of the newly synthesized DNA strands were then selectively tagged with a second specific sequence tag to produce di-tagged DNA molecules with known sequence tags at 5’ and 3’ ends. Di-tagged molecules were then enriched by PCR with unique indexed PCR primers to provide dsDNA libraries with Illumina sequencing adapters that could be multiplexed and sequenced on a single flow cell. For all other WGBS experiments the Accel-NGS Methyl-Seq DNA Library Kit (Swift BioSciences) was used. For the Swift kit, bisfulfite converted single-stranded DNA was first denatured before simultaneous tailing and ligation of truncated adapters to 3’ ends with Adaptase. An extension step then incorporates the truncated adapter 1 by a primer extension reaction, before a ligation step adds truncated adapter 2 to the bottom strand only. 9 cycles of PCR were used to increase yield and incorporate full-length adapters for unique dual-indexed sequencing. Finally, Agencourt AMPure XP beads (Beckman Coulter) were used to remove oligonucleotides and small fragments and change enzymatic buffer composition. Libraries were quantified using the Qubit dsDNA HS assay kit, assessed for size and quality using the Agilent Bioanalyser with the DNA HS Kit and combined in a single equimolar pool. Sequencing was performed on a P2 flow cell on the Illumina NextSeq 2000 platform using NextSeq 1000/2000 P2 Reagents v3 kit (200 Cycles), or on the NextSeq 550 platform using NextSeq 500/550 High-Output v2 kit (150 cycles). PhiX Control v3 Library was spiked in at a concentration of ~5% to increase library diversity for sequencing. Library preparation and sequencing was performed by the Edinburgh Clinical Research Facility.
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| Library strategy |
Bisulfite-Seq |
| Library source |
genomic |
| Library selection |
RANDOM |
| Instrument model |
NextSeq 550 |
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| Data processing |
Whole Genome Bisulfite Sequencing (WGBS):
Sequencing quality was assessed with FASTQC (v0.11.4). Low quality reads and remaining adaptors were removed using TrimGalore (v0.4.1, Settings: --adapter AGATCGGAAGAGC --adapter2 AAATCAAAAAAAC). The paired end reads were then aligned to the hg38 genome using Bismark (v 0.18.1 with Bowtie2 v2.3.1 and settings: -N 0 -L 20, trimming settings --clip_r1 10 --clip_r2 13 --three_prime_clip_r1 1 --three_prime_clip_r2 1) (Langmead and Salzberg, 2012, PMID: 22388286, Krueger and Andrews, 2011, PMID: 21493656 ) before PCR duplicates were identified and removed using Bismark’s deduplicate_bismark command. Aligned BAM files were processed to report coverage and number of methylated reads for each CpG observed. Forward and reverse strands were combined using Bismark’s methylation extractor and bismark2bedgraph modules with custom Python and AWK scripts. Processed WGBS files were assessed for conversion efficiency based on the proportion of methylated reads mapping to the phage-λ genome spike-in (>99.5% in all cases). BigWigs for visualisation of WGBS data were generated using the coverage in 2.5 kb-sized windows across the genome. These were defined using BEDtools (v2.27.1) (Quinlan and Hall, 2010, PMID: 20110278) and coverage and converted to bigWigs using UCSC tools bedGraphToBigWig (v326). Windows with a total coverage <10 were excluded before conversion to bigWigs.
Assembly: hg38
Supplementary files format and content: Processed coverage values for each CpG observed in sample (.cov files). CpG position and total coverage (T) and methylated coverage (M) combining both strands. Columns = chr, start, end, T, M. Positions are 0-based, ie BED format.
Supplementary files format and content: bigWig files for each sample also supplied. Mean methylation in 2.5Kb windows across genome. Only windows with coverage >= 10 in that sample are in each file.
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| Submission date |
Oct 03, 2023 |
| Last update date |
Jan 22, 2024 |
| Contact name |
Duncan Sproul |
| Organization name |
University of Edinburgh
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| Department |
MRC Human Genetics Unit
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| Street address |
Crewe Road South
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| City |
Edinburgh |
| State/province |
Mid Lothian |
| ZIP/Postal code |
EH4 2XU |
| Country |
United Kingdom |
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| Platform ID |
GPL21697 |
| Series (2) |
| GSE244517 |
DNMT3B PWWP mutations cause hypermethylation of heterochromatin (WGBS) |
| GSE244520 |
DNMT3B PWWP mutations cause hypermethylation of heterochromatin. |
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| Relations |
| BioSample |
SAMN37657528 |
| SRA |
SRX21974121 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM7818549_3BKO_3Bw263a_exp_2_WGBS.cov.gz |
119.6 Mb |
(ftp)(http) |
COV |
| GSM7818549_3BKO_3Bw263a_exp_2_WGBS_2500bp_win.bigWig |
8.6 Mb |
(ftp)(http) |
BIGWIG |
SRA Run Selector |
| Raw data are available in SRA |
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