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Sample GSM7135347 Query DataSets for GSM7135347
Status Public on Jun 30, 2023
Title HEPG2 input for FLAG-TDG-N140A-CD and MBD3
Sample type SRA
 
Source name HepG2
Organism Homo sapiens
Characteristics cell line: HepG2
cell type: Hepatocellular carcinoma
treatment: stably expressing 3XFLAG-TDG-N140A-CD
chip antibody: none
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 SAMN34035749
SRA SRX19843155

Supplementary data files not provided
SRA Run SelectorHelp
Raw data are available in SRA
Processed data not provided for this record

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