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Sample GSM6156757 Query DataSets for GSM6156757
Status Public on Jan 30, 2023
Title ChIP_k4me1_CO_679
Sample type SRA
 
Source name Colon
Organism Canis lupus familiaris
Characteristics tissue: Colon
Sex: Female
breed: Beagle
chip antibody: H3K4me1 (Abcam, #ab8895)
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 The 11 frozen tissues were thawed on ice and 10 mg of tissue per IP reaction was chopped into ~1 mm3 pieces with two razor blades on the ice. Chopped tissues were washed out with PBS buffer containing Protease K, 10mM PMSF, and 10mM Sodium Butyrate histone deacetylase inhibitor to remove blood from the tissue. Washed tissue was grinded using pestle and mortar. Then prepared cell mass was cross-linked in the PBS buffer with 1.5% Formaldehyde added at room temperature(RT) for 20 minutes. By adding 125 mM Glycine and placing the samples on the rotator for 5 min, cross-linking reactions were stopped. Fixed cell mass was washed twice with PBS buffer containing Protease K, 10mM PMSF, and 10mM Sodium Butyrate. Fixed cell mass was lysed using buffer A (5 mM PIPES buffer, 85 mM KCl, and 0.5% NP 40). The supernatant was centrifuged out and added to buffer B (50 mM Tris-HCl, 0.5% SDS, and 2.5mM EDTA). All buffers contained Protease K, 10 mM PMSF, and 10 mM Sodium Butyrate histone deacetylase inhibitor. Sonication was then carried out to get 300-500 bp size fragments using Bioruptor Pico (Diagenode). Twenty to fifty cycles of 30 seconds, on and off, were performed at 4 ℃ depending on the tissue. Liver cells were sonicated for only 20 cycles. Chromatin solution was centrifuged to remove the debris and diluted with ChIP IP buffer (16.7 mM Tris-HCl, 0.05% SDS, 1.1% Triton X-100, 1.2 mM EDTA, and 167 mM NaCl). After adding 5 µg of sample to 10 µg of anti-H3K4me1 (ab8895), H3K27Ac (ab4729), H3K4me3 (ab8580), H3K27me3 (ab6002), H3K9me3 (ab8898), and IgG (Sc-2027) per IP reaction, the chromatin solution was incubated overnight at 4 ℃. The cross-linking was reversed and DNA was purified after treatment with protease K and RNase A. ChIPed DNA was quantified using the Qubit fluoro-spectrometer (ThermoFisher Scientific, Waltham, MA, USA) and the enrichment was validated by PCR.
The ChIP library was prepared using the TruSeq ChIP Library Preparation Kit and sequenced in ~500 bp fragments using HiSeq 2500 system (Illumina) by Macrogen (Macrogen, Korea).
 
Library strategy ChIP-Seq
Library source genomic
Library selection ChIP
Instrument model Illumina NovaSeq 6000
 
Description ChIP_CO_k4me1_optimal.narrowPeak
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)
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
Street address 1, Gwanak-ro, Gwanak-gu
City Seoul
ZIP/Postal code 08826
Country South Korea
 
Platform ID GPL25760
Series (2)
GSE203104 Integrated mapping of the dog epigenome (ChIP-seq)
GSE203107 Integrated mapping of the dog epigenome
Relations
BioSample SAMN28451666
SRA SRX15289009

Supplementary data files not provided
SRA Run SelectorHelp
Raw data are available in SRA
Processed data are available on Series record

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