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Sample GSM7433451

Query DataSets for GSM7433451

Status

Public on May 31, 2023

Title

Colon (validation), 3 Months, Rep1

Sample type

SRA

Source name

Colon

Organism

Mus musculus

Characteristics

tissue: Colon
strain: C57BL6/J
Sex: Male
age: 3 Months

Growth protocol

All the mice were kept solely for aging in a controlled environment and health status.

Extracted molecule

genomic DNA

Extraction protocol

Genomic DNA was isolated with the DNeasy Blood & Tissue Kit (Qiagen).
200 ng of DNA was fragmented via a 5 hour Msp1-digest (New England Biolabs), followed by TrueSeq adapter ligation (Illumina, San Diego, CA, USA) and subsequent bisulfite conversion following the manufacturers protocol (EZ DNA Methylation Gold Kit, Zymo Research). The bisulfite converted DNA was amplified in a 19-cycles PCR with Pfu Turbo Cx Hotstart DNA Polymerase (Agilent Technologies) and the resulting libraries were quality checked using 4200 TapeStation (Agilent Technologies). For sequencing of validation samples, Illumina’s next-generation sequencing methodology 87 was used. In detail, genomic DNA was quality checked and quantified using the 4200 TapeStation instruments in combination with the Genomic DNA ScreenTape (both Agilent Technologies). Libraries were prepared from 100 ng of input material using Ovation RRBS Methyl-Seq with TrueMethyl oxBS (Tecan). In detail, only the bisulfite part of the protocol was followed, oxidation of DNA was not done, and samples were treated as MOCK oxBS samples according to the manufacturer’s instruction. Quantification and quality check of libraries were done using the 4200 TapeStation and D5000 ScreenTapes instruments (both Agilent Technologies).

Library strategy

Bisulfite-Seq

Library source

genomic

Library selection

Reduced Representation

Instrument model

Illumina NovaSeq 6000

Description

3M.N1
colon_validation_CpG_methylationrates.bedg.gz
colon_validation_CpG_coverages.bedg.gz

Data processing

Sequence information was converted to fastq format using bcl2fastq v2.20.0.422. From validation samples only those reads were downstream processed, that start with a Tecan/NuGen TrueMethyl diversity adapter (length 0-3, clipped afterwards) followed by a MspI cutting site.
Read quality trimming (cutoff 20) and TruSeq universal adapter clipping was performed using Cutadapt v2.10. Due to MspI cutting site end-repair, R1 adapter was prefixed by NN and in case of paired-end data, unconditional 5' clipping of length 2 was conducted on R2.
Mappings were done via segemehl v0.3.4 (methyl-C mode, accuracy 95%, reference GRCm38) and filtered for unique alignments. Paired-end alignments were further filtered for proper-read pairs and overlaps of R2 with R1 were trimmed using BamUtil clipOverlap v1.0.14.
Validation data alignments were deduplicated for over amplified PCR fragments based on unique molecular identifiers utilizing UMI-tools v1.1.1.
Methylation rates were inferred utilizing haarz v0.3.0. Rates and coverages within a CpG context were compiled using BEDTools v2.30.0.
Assembly: mm10
Supplementary files format and content: Tabular delimited text files in bedg format of read alignment coverages and inferred methylation rates per GRCh38 chromosomal CpG and sample.

Submission date

May 30, 2023

Last update date

May 31, 2023

Contact name

Konstantin Riege

E-mail(s)

konstantin.riege@leibniz-fli.de

Organization name

Leibniz Institute on Aging - Fritz Lipmann Institute (FLI)

Street address

Beutenbergstraße 11