|
| Status |
Public on Jun 30, 2023 |
| Title |
APOBEC-sequencing of ChIP DNA for FLAG-TDG-N140A-CD, replicate 1 |
| Sample type |
SRA |
| |
|
| Source name |
HEK293
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: HEK293
cell type: Human embryonic kidney
treatment: stably expressing 3XFLAG-TDG-N140A-CD
chip antibody: M2 Monoclonal ANTI-FLAG (Sigma, F1804)
|
| Treatment protocol |
Cells were stably expressing 3XFLAG-TDG-N140A-CD (for FLAG ChIP) from a lentiviral vector.
|
| Growth protocol |
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 200 ng fragmented input or 3.5-200 ng ChIP DNA using the NEBNext® Enzymatic Methyl-seq Kit (NEB, cat. no. E7120L) with some modifications to the manufacturer’s protocol. End prep and ligation of EM-seq adapter ligation were performed as per the manufacturer’s protocol except that the NEBNext EM-seq Adapter was substituted with 5hmC Adapter provided as a gift by NEB: this is critical as methylated NEBNext Em-seq Adapters included with the kit are not resistant to APOBEC-conversion alone. Cleanup after adapter ligation was performed with NEBNext Sample Purification Beads as per manufacturer’s protocol but elution volume was reduced to 17 µL for compatibility with the modified protocol. The TET2 oxidation step of the NEBNext® Enzymatic Methyl-seq Kit protocol was then skipped and 16 µL eluant was used directly for the APOBEC conversion (denaturation and deamination) steps. Denaturation with formamide, deamination with APOBEC, cleanup, PCR amplification, and final cleanup steps were performed according to the manufacturer’s protocol. Libraries were quantified using the KAPA Library Quanitification Kits - Complete kit (Universal) (Kapa Biosystems). Average size fragment was determined using a LabChip GX (PerkinElmer) instrument. The libraries were normalized and pooled and then denatured in 0.05N NaOH and neutralized using HT1 buffer. The pool was loaded at 200pM on a Illumina NovaSeq S4 lane using Xp protocol as per the manufacturer’s recommendations. The run was performed for 2x150 cycles (HEK293 cells) or 2x100 cycles (mouse cortices) (paired-end mode). A phiX library was used as a control and mixed with libraries at 1% level.
|
| |
|
| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina NovaSeq 6000 |
| |
|
| Data processing |
Base calling was performed with RTA v3.4.4.
The 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.
Reads were aligned to custom HEK293 (GRCh38) reference genomes using bismark v0.23.1 with default parameters. Custom HEK293 reference genome has the same chromosomal coordinates as GRCh38 and was built by merging all (non-converted) ChIP-seq alignments (associated with this dataset) from anti-FLAG-TDG/input samples, calling SNPs and INDELs with freebayes v1.3.6, filtering for SNPs with QUAL scores above 30 using vcffilter v1.0.3, and applying called SNPs (no INDELs) to the hg38 genome using the Genome Analysis Toolkit (GATK) v4.2.6.1 FastaAlternateReferenceMaker tool.
Bismark alignments were processed by Bismark deduplication and methylation extractor scripts.
Assembly: GRCh38
Supplementary files format and content: Bismark deduplicated coverage files.
Library strategy: APOBEC-seq
|
| |
|
| 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) |
| GSE228703 |
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 APOBEC-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 |
SAMN34035670 |
| SRA |
SRX19843235 |
