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| Status |
Public on Jun 30, 2023 |
| Title |
APOBEC-seq of input DNA from adult mouse cortex animal no. 5 |
| Sample type |
SRA |
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| Source name |
cortex
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| Organism |
Mus musculus |
| Characteristics |
animal: animal no. 5
tissue: cortex
strain: C57BL/6J
Sex: female
age: 14 weeks
chip antibody: none
genotype: wild-type
treatment: APOBEC conversion of DNA
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| Growth protocol |
Surgically excised cerebral cortices of wild-type adult (14 weeks) C57BL/6J female mice were purchased from the Jackson Laboratory and shipped frozen.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Chromatin immunoprecipitation of mouse cortices was performed with the ChIP-IT High Sensitivity® Kit (Active Motif, cat. no 53040) according to the manufacturer’s protocol. Frozen mouse cortices were cut into ~1 mm cubes in 10 mL Complete Tissue Fixation Solution in a 100-mm petri dish and incubated, with rotation, at room temperature for 15. The reaction was quenched with 515 µL Stop Solution followed by incubation at room temperature for 5 minutes. The cross-linked tissues were then homogenized by passing the suspension 20 times through a 20-guage needle attached to a sterile plastic syringe. The tissues were pelleted by centrifugation at 1,250 x g for 3 min at 4 °C and pellets were washed twice with 10 mL ice-cold PBS Wash Buffer. Washed pellets were resuspended in 5 mL Chromatin Prep Buffer supplemented with 5 µL protease inhibitor cocktail (PIC) and 5 µL 100 µM PMSF, incubated on ice for 10 minutes, and homogenized again to lyse cell membranes. Nuclei were pelleted by centrifugation as before, resuspended in 1 mL ChIP buffer, incubated for 10 min on ice, and sonicated in a Bioruptor (Diagenode) sonicator in 200 µL aliquots, each receiving 6 10-min rounds of sonication at high power (30 sec on / 30 sec off). Sonicated samples were clarified by centrifugation for 2 minutes at 4 °C at 16,000 x g and stored at -80 °C. Input DNA was prepared as follows: 25 µL was removed and purified according to the manufacturer’s protocol (briefly, RNAse A treatment, Proteinase K treatment, addition of NaCl and reverse cross-linking at 65 °C for 16 hours, and purification by phenol:chloroform and ethanol extraction) and quantified using a NanoDrop spectrophotometer. 30 µg sheared chromatin was immunoprecipitated overnight at 4 °C by dilution in 200 µL ChIP Buffer supplemented with 5 µL PIC and incubation with 4 µg antibody with 5 µL Blocker. 30 µL washed Protein G agarose beads were added to the reaction and incubated for 3 h. The reactions were then diluted by the addition of 600 µL ChIP buffer and added to ChIP filtration columns, washed 5 times with Wash Buffer AM1, and eluted in 100 µL Elution Buffer AM4. ChIP DNA was purified in the same manner as input DNA and in 36 µL elution volume for ChIP-sequencing.
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.
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina NovaSeq 6000 |
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| 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 mouse (GRCm39) reference genomes using bismark v0.23.1 with default parameters.
Bismark alignments were processed by Bismark deduplication and methylation extractor scripts.
Assembly: GRCm39
Supplementary files format and content: Bismark deduplicated coverage files.
Library strategy: APOBEC-seq
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| Submission date |
Apr 01, 2023 |
| Last update date |
Jun 30, 2023 |
| Contact name |
Moshe Szyf |
| E-mail(s) |
moshe.szyf@mcgill.ca
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| Phone |
5143987107
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| Organization name |
McGill University
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| Department |
Pharmacology & Therapeutics
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| Street address |
3655 Promenade Sir William Osler, Room 1309
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| City |
Montreal |
| State/province |
QC Quebec |
| ZIP/Postal code |
H3G1Y6 |
| Country |
Canada |
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| Platform ID |
GPL24247 |
| Series (2) |
| GSE228705 |
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 [mouse 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 |
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| Relations |
| BioSample |
SAMN34035799 |
| SRA |
SRX19843214 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM7135368_NS.1990.002.NEBNext_EM-seq_i7_22---NEBNext_EM-seq_i5_22.cortex_5_input_mm39_bismark_bt2_pe.deduplicated.bismark.cov.gz |
53.8 Mb |
(ftp)(http) |
COV |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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