|
| Status |
Public on Jun 30, 2023 |
| Title |
HEPG2 anti-MBD3 |
| Sample type |
SRA |
| |
|
| Source name |
HepG2
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: HepG2
cell type: Hepatocellular carcinoma
treatment: endogenous TDG
chip antibody: MBD3 (abcam, ab157464)
|
| Treatment protocol |
Cells were untreated (for MBD3 ChIP) or stably expressing 3XFLAG-TDG-N140A-CD (for FLAG ChIP) from a lentiviral vector.
|
| Growth protocol |
All cell lines were maintained in DMEM, high glucose (Thermo Fisher, cat. no. 11965118) supplemented with 10% fetal bovine serum (FBS) (Wisent, cat. no. 080-150) and 1X Penicillin-Streptomycin-Glutamine (Thermo Fisher, cat. no. 10378016) and grown in a 37 °C humidifed 5% CO2 incubator. Media was replaced or cells were passaged every 3 days.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
150mm tissue culture dishes containing 90% confluent cells from each experimental condition were cross-linked and immunoprecipitated with the Active Motif ChIP-IT High Sensitivity Kit according to manufacturers instructions and using indicated antibodies. Samples were sonicated on the Bioruptor (Diagenode) in 1.5 mL Eppendorf tubes at high power for two 10-minute cycles of 30 seconds on and 30 seconds off, replacing warmed water with ice-cold water and minimal ice between each cycle. 25 µL of sonicated chromatin was purified according to ChIP-IT HS Kit protocol and quantified by NanoDrop. 30 µg input chromatin was used for immunoprecipitation.
Libraries were generated from 25 µL of fragmented DNA (range 100-300 bp) using the NEBNext Ultra II DNA Library Prep Kit for Illumina (NEB), as per the manufacturer’s recommendations. Adapters and PCR primers were purchased from Integrated DNA Technologies (IDT). Size selection was carried out using SparQ beads (QIAGEN) prior to PCR amplification (12 cycles). Libraries were quantified using the KAPA Library Quanitification Kits - Complete kit (Universal) (Kapa Biosystems). Average fragment size was determined using a LabChip GX (PerkinElmer) instrument. The libraries were normalized, pooled, and then denatured in 0.05 N NaOH and neutralized using HT1 buffer. The pool was loaded at 175 pM (HEK293 cells) or 200pM (mouse cortices) on a Illumina NovaSeq S4 lane using Xp protocol as per the manufacturer’s recommendations. The run was performed for 2x100 cycles (paired-end mode). A phiX library was used as a control and mixed with libraries at 1% level.
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina NovaSeq 6000 |
| |
|
| Data processing |
Base calling was performed with RTA v3.4.4 . Program bcl2fastq2 v2.20 was then used to demultiplex samples and generate fastq reads.
Paired-end FASTQ files were trimmed for quality and adapter content using Trim Galore v0.6.7 with default parameters.
Trimmed FASTQ files were aligned to hg38 reference genome using bowtie239 v2.4.5 with default parameters.
SAM files were converted to BAM format and unmapped reads were removed with samtools v1.16.1
BAM files were sorted and duplicates were marked with Picard tools v2.18.29 and indexed with samtools.
Significant peaks were identified with the callpeak function from macs2 v2.2.7.1 using the respective input sequencing data for each sample as control and precompiled values for the mappable human genome size.
Assembly: hg38
Supplementary files format and content: narrowPeak (except for input samples)
|
| |
|
| Submission date |
Apr 01, 2023 |
| Last update date |
Jun 30, 2023 |
| Contact name |
Moshe Szyf |
| E-mail(s) |
moshe.szyf@mcgill.ca
|
| Phone |
5143987107
|
| Organization name |
McGill University
|
| Department |
Pharmacology & Therapeutics
|
| Street address |
3655 Promenade Sir William Osler, Room 1309
|
| City |
Montreal |
| State/province |
QC Quebec |
| ZIP/Postal code |
H3G1Y6 |
| Country |
Canada |
| |
|
| Platform ID |
GPL24676 |
| Series (2) |
| GSE228704 |
Bisulfite-free base-resolution sequencing of oxidized cytosines (APOBEC-seq) reveals a ubiquitous role of thymine DNA glycosylase in active gene promoters and an interaction with MBD3/NuRD [human ChIP-seq] |
| GSE228707 |
Bisulfite-free base-resolution sequencing of oxidized cytosines (APOBEC-seq) reveals a ubiquitous role of thymine DNA glycosylase in active gene promoters and an interaction with MBD3/NuRD |
|
| Relations |
| BioSample |
SAMN34035740 |
| SRA |
SRX19843164 |
