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Sample GSM6156889

Query DataSets for GSM6156889

Status

Public on Jan 30, 2023

Title

MBD_input_LI_jj8

Sample type

SRA

Source name

Liver

Organism

Canis lupus familiaris

Characteristics

tissue: Liver
Sex: Female
breed: Beagle

Treatment protocol

Three dogs in healthy condition were euthanized by intravenous administration of alfaxalone and KCl. Eleven common tissues (cerebrum [CR], cerebellum [CL], colon [CO], kidney [KI], liver [LI], lung [LU], mammary gland [MG], ovary [OV], pancreas [PA], spleen [SP] and stomach [ST]) were isolated from the three beagles. All tissues were rinsed with ice-cold PBS before being minced. Immediately after mincing, tissues were divided into 4 tubes based on the types of analysis, and frozen with liquid nitrogen

Growth protocol

One male and two female beagles were kindly donated by Dr. Jong-Gu Kang from the College of Veterinary Medicine at Chungbuk National University.

Extracted molecule

genomic DNA

Extraction protocol

Beagle tissues (11 types, ~25mg each) were incubated in Buffer ATL (Qiagen, USA) and protease K at 56°C until the tissue was completely lysed. Genomic DNA was extracted using the DNeasy Blood and Tissue Kit (Qiagen, USA), according to the manufacturer's protocol with modifications. The concentration and purity of isolated genomic DNA was assessed by NanoDrop. For each sample, 3 μg of DNA, normalized to 20 ng/μl, was subjected to shearing with the Bioruptor® Pico (Diagenode) and the size of fragmented DNA (~300 bp) was verified on an agarose gel. Finally, the concentration of sheared double-stranded DNA was measured using Qubit dsDNA HS Assay (ThermoFisher Scientific, USA). For MBD-enrichment, 500 ng of fragmented double-stranded DNA was enrichedwith methylated CpGs using MethylMiner™ Kit (Invitrogen, cat# ME10025), according to the manufacturer's instructions. To counteract the possibility of increased unspecific binding, we used more stringent wash conditions. In more detail, for each capture reaction, prepared MBD-beads (10 μg beads and 350 ng MBD-biotin protein were used in the same reaction) were added to each 500 ng of fragmented DNA input. Prepared MBD-beads were diluted to 1x in Bind/Wash Buffer prior to addition of each DNA sample, to increase pipetting accuracy. Each capture reaction was brought up to 200 μl final volume in 1x Bind/Wash Buffer, and incubated on a rotator for 40 min at RT. Following incubation, each tube was placed on a magnet for 1 minute and the supernatant containing the non-captured (unmethylated) DNA fragments was removed. The beads with bound methylated DNA were then washed twice by incubation with 200 μl Bind/Wash Buffer. The beads were washed with Bind/Wash buffer, according to the protocol, and eluted in step-wise elution using 200 μl of serially diluted elution buffer (200, 300, 400, 600, and 800 mM).
Eluted methylated DNA fragments in the 600 and 800 mM elution buffers underwent cluster generation and 200 bp paired-end sequencing using TruSeq Nano DNA Kit on the Illumina HiSeq 4000 instrument (Illumina, San Diego, CA, USA).

Library strategy

MBD-Seq

Library source

genomic

Library selection

MBD2 protein methyl-CpG binding domain

Instrument model

Illumina HiSeq 4000

Data processing

All of pre-processing steps including trimming, alignment, generation of signal track, peak calling and diverse type of QC were performed using ENCODE ChIP-seq processing pipeline (https://github.com/ENCODE-DCC/chip-seq-pipeline2)
Fold-change in each 100bp bins were calculated using MEDIPS to identify methyl-CpG binding domain enriched sites compared to corresponding input samples in 11 differenct canine normal tissues
Assembly: canFam3.1
Supplementary files format and content: narrowPeak (except for Input sample)

Submission date

May 16, 2022

Last update date

Jan 30, 2023

Contact name

Je-Yoel Cho

E-mail(s)

jeycho@snu.ac.kr

Organization name

Seoul National University

Department

Biochemistry