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Sample GSM1104076

Query DataSets for GSM1104076

Status

Public on Apr 18, 2013

Title

5fC enriched DNA from Tdg fl/fl mouse EB cells, replicate 1

Sample type

SRA

Source name

5fC enriched DNA from Tdg fl/fl mouse EB cells

Organism

Mus musculus

Characteristics

strain: C57BL/6-ICR
cell type: Mouse ES cells differentiated to embryoid body (EB)

Treatment protocol

EB differentiation was induced in suspension in 10 cm bacterial dishes in medium without LIF, CHIR99021 and PD0325901. Change half of the medium every day for 5 days before harvesting mEBs by sedimentation

Growth protocol

mESCs were cultured in feeder-free gelatin-coated plates in Dulbecco’s Modified Eagle Medium (DMEM) (Invitrogen Cat. No. 11995) supplemented with 15% FBS (GIBCO), 2 mM L-glutamine (GIBCO), 0.1 mM 2-mercaptoethanol (Sigma), 1×nonessential amino acids (GIBCO), 1,000 units/ml LIF (Millipore Cat. No. ESG1107), 1×pen/strep (GIBCO), 3 mM CHIR99021 (Stemgent), and 1 mM PD0325901 (Stemgent).

Extracted molecule

genomic DNA

Extraction protocol

25 ng of 5fC-enriched DNA, 5hmC-enriched DNA, or non-enriched sonicated input genomic DNA was end-repaired, adenylated, and ligated to Illumina Genomic DNA Adapters (Genomic DNA adapter oligo mix) according to standard Illumina protocols for ChIP-Seq library construction, maintaining the proper molar ratios of adapter to insert. Adapter-ligated fragments of ~200-350 bp were gel-purified by 2% agarose gel electrophoresis and PCR-amplified for 18 PCR cycles. Libraries were checked for quality and quantified using an Agilent 2100 Bioanalyzer DNA 1000 Chip.
300 ng of H3K4me1 ChIP'd DNA (Abcam, ab8895) was subjected to fCAB treatment and subsequently bisulfite converted in parallel with untreated ChIP’d DNA, followed by end-repair, adenylation and ligation to methylated Illumina TruSeq DNA adapters according to the Illlumina Methyl-Seq Protocol

Library strategy

OTHER

Library source

genomic

Library selection

other

Instrument model

Illumina HiScanSQ

Description

processed data file: Tdg.WT.EB.5fC.regions.bed

Data processing

library strategy: ChIP-Seq type, for Selective chemical labeling and enrichment of 5fC or 5hmC containing DNA fragments
Illumina Basecalling, CASAVA 1.8.2
FASTQ sequences were aligned to NCBIv1/mm9 with Bowtie v0.17.2 retaining unique non-duplicate matches to the genome, with no more than 3 mismatches in the first 30 bases
Enriched regions derived from MACS v1.4, --bw=200 -g 1.87e9, using genotype matched input controls
library strategy: ChIP-Methyl-Seq or ChIP-fCAB-Seq
H3K4me1-ChIP-Methyl-Seq and H3K4me1-ChIP-fCAB-Seq paired-end reads (fastq) were first pre-processed to remove adapter sequences, as well as low quality sequence on both the 3' and 5' ends using Trimmomatic 0.20, with the following parameters: LEADING:3 TRAILING:3 SLIDINGWINDOW:4:15 MINLEN:36. This was followed by in silico conversion of each C to T (Read 1) and each G to A (Read 2). Pre-processed reads were then aligned to both C to T and G to A converted chromosomes that were computationally derived from NCBI mm9 genomic sequence using Bowtie 0.12.9 (-m 1 -l 30 -n 0 -e 90 -X 550). Reads mapping to both genomes are discarded and non-aligned pairs were re-processed as single-end data with Bowtie 0.12.9 (-m 1 -l 30 -n 0 -e 90. For both paired-end and single alignments, only uniquely mapping reads were retained and PCR duplicates were removed using MarkDuplicates (Picard Tools 1.82). To avoid counting reference positions covered by overlapping paired-end reads, overlapping regions were clipped, keeping the region of the overlap with higher quality. The original computationally converted Cs and Gs were reverted, and for each reference cytosine position the number C reads and T reads were counted using SAMTools mpileup.
Genome_build: mm9
Supplementary_files_format_and_content: BED format, containing regions of enrichment

Submission date

Mar 22, 2013

Last update date

May 15, 2019

Contact name

Keith E Szulwach

E-mail(s)

kszulwa@emory.edu

Phone

404-712-0796

Organization name

Emory University