|
| Status |
Public on Nov 24, 2020 |
| Title |
HCT116_aza_treated_day_3 |
| Sample type |
SRA |
| |
|
| Source name |
Human cell line
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: HCT116
treatment: 5-aza-2-deoxycytidine
timepoint (days): 3
genotype: HCT116 cells
|
| Treatment protocol |
Cells were plated at ~20% confluency. Next day cells were treated with freshly prepared 1µM 5-aza-dC (Sigma Aldrich) for 24 hrs. Cell pellets were collected at different time points following treatment and genomic DNA was extracted using DNeasy Blood & Tissue Kit (QIAGEN) following the manufacturer’s instructions.
|
| Growth protocol |
HCT116, DNMT1 KO, DNMT3B KO, and DKO 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.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Genomic DNA was extracted from cells using DNeasy Blood & Tissue Kit according to the manufacturer’s protocol with some modifications. RNase A/T1 Cocktail (Ambion AM2286) was added to proteinase K and samples were incubated at 37°C for 1hr to remove RNA. At the final step, DNA was eluted with ddH2O instead of AE buffer. DNA was quantified by Nanodrop and Qubit.
200ng of purified DNA samples were processed using the Ovation RRBS Methyl-Seq system kit (NuGen Technologies) according to instructions with modifications. Briefly, 0.5ng unmethylated phage λ DNA (NEB) was spiked into each sample to allow assessment of bisulfite conversion efficiency. The methylation-insensitive restriction enzyme MspI was then used to digest the genomic DNA, and digested fragments were ligated to adapters. Adapter-ligated fragments were then repaired before bisulfite conversion with the Qiagen Epitect Fast Bisulfite Conversion kit. Bisulfite-treated adapter-ligated fragments were amplified by 9 cycles of PCR and purified using Agencourt RNAClean XP beads. Libraries were quantified using the Qubit dsDNA HS assay and assessed for size and quality using the Agilent Bioanalyzer DNA HS kit. Library preparation and sequencing was performed by the Edinburgh Clinical Research Facility.
|
| |
|
| Library strategy |
Bisulfite-Seq |
| Library source |
genomic |
| Library selection |
Reduced Representation |
| Instrument model |
Illumina NextSeq 500 |
| |
|
| Description |
HCT116_aza_d3
|
| Data processing |
Raw Illumina sequencing output from the NextSeq were converted to paired FASTQ files without demultiplexing using ‘bcl2fastq’ and default settings (v2.17.1.14). These FASTQ files were then demultiplexed using custom python scripts considering indexes with perfect matches to the sample indexes. The different lanes for each sample were then combined.
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). NuGen adaptors contain extra diversity bases to facilitate sequencing. These were removed using the ‘trimRRBSdiversityAdaptCustomers.py’ Python script provided by NuGen (v1.11). The paired end reads were then aligned to the mm10 genome using Bismark (v 0.16.3 with Bowtie2 v2.2.6 with settings: -N 0 -L 20) before PCR duplicates were identified and removed using the 6bp UMIs present in the index reads and the ‘nudup.py’ Python script supplied by NuGen (v2.3). 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 RRBS files were assessed for conversion efficiency based on the proportion of methylated reads mapping to the λ genome spike-in (>99.5% in all cases).
BigWigs were generated from RRBS data using CpGs with coverage ≥5
Genome_build: hg38
Supplementary_files_format_and_content: Processed coverage values for each CpG observed in sample. 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. Only contain CpGs with coverage >= 5 in that sample.
|
| |
|
| Submission date |
Sep 20, 2019 |
| Last update date |
Nov 24, 2020 |
| Contact name |
Duncan Sproul |
| Organization name |
University of Edinburgh
|
| Department |
MRC Human Genetics Unit
|
| Street address |
Crewe Road South
|
| City |
Edinburgh |
| State/province |
Mid Lothian |
| ZIP/Postal code |
EH4 2XU |
| Country |
United Kingdom |
| |
|
| Platform ID |
GPL18573 |
| Series (2) |
| GSE137812 |
De novo DNA methyltransferase activity in colorectal cancer is directed towards H3K36me3 marked CpG island (RRBS) |
| GSE158406 |
De novo DNA methyltransferase activity in colorectal cancer is directed towards H3K36me3 marked CpG island |
|
| Relations |
| BioSample |
SAMN12802279 |
| SRA |
SRX6882173 |
