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Sample GSM6156841

Query DataSets for GSM6156841

Status

Public on Jan 30, 2023

Title

ChIP_input_CL_679

Sample type

SRA

Source name

Cerebellum

Organism

Canis lupus familiaris

Characteristics

tissue: Cerebellum
Sex: Female
breed: Beagle
chip antibody: none

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

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