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Sample GSM2465050 Query DataSets for GSM2465050
Status Public on Jan 24, 2017
Title H3K27me3_ChIPSeq_siHP1γ
Sample type SRA
Source name mouse stem cells E14
Organism Mus musculus
Characteristics Sex: male
cell type: embryonic stem cell line derived from mouse inner-cell masses
stain: 12910la
chip antibody: H3K27me3 (Upstate, 07-449, lot DAM1514011)
trasfection: tranfected with siHP1g for 48h
Treatment protocol Transfection of ES cells was performed by electroporation with NeonTM transfection system (Invitrogen). Basically, 5×106 trypsinized cells were washed once with PBS, and resuspended in 90 μl Buffer R. The single cell suspension was then gently mixed with siRNA (20 μM) and subjected to electroporation (1400 V/10 ms/3 pulse). Cells were subsequently seeded in a 10 cm dish, and cultured for additional 48h before harvesting for further experiments.
Growth protocol Embryonic stem cell lines were cultured in KnockoutTM DMEM (Life Technology) supplemented with 15% FBS (Clonetech), 1% Non-essential amino acids, 1% L-Glutamine, 10-4 M β-Mercaptoethanol and 103 U/ml LIF.
Extracted molecule genomic DNA
Extraction protocol Lysates were clarified from sonicated nuclei and histone-DNA complexes were isolated with antibody.
Libraries were prepared according toThruPLEX® DNA-seq Kit Instruction Manual (CAT. NO. R400406). Briefly, DNA was end-repaired using a combination of T4 DNA polymerase, E. coli DNA Pol I large fragment (Klenow polymerase) and T4 polynucleotide kinase. The blunt, phosphorylated ends were treated with Klenow fragment (32 to 52 exo minus) and dATP to yield a protruding 3- 'A' base for ligation of Illumina's adapters which have a single 'T' base overhang at the 3’ end. After adapter ligation DNA was PCR amplified with Illumina primers for 15 cycles and library fragments of ~250 bp (insert plus adaptor and PCR primer sequences) were band isolated from an agarose gel. The purified DNA was captured on an Illumina flow cell for cluster generation. Libraries were sequenced on the Genome Analyzer following the manufacturer's protocols.
Library strategy ChIP-Seq
Library source genomic
Library selection ChIP
Instrument model HiSeq X Ten
Data processing Basecalls performed using FastQC v0.11.2
ChIP-seq reads were aligned to the mm9 genome assembly using bowtie2 version 2.3.3 with the follow setting: -p 8
Data were filtered using trim galore version 0.3.7, and the following specifications:-q 25 --stringency 5 --length 50
peaks were called using MACS version 1.4.2 with the following setting: -g mm -m 10, 30 -p 10e-5 --nomodel --shiftsize 200 -w
Genome_build: mm9
Supplementary_files_format_and_content: Bigwig files were generated using wigToBigWig with default setting.
Submission date Jan 23, 2017
Last update date May 15, 2019
Contact name Pinchao Mei
Phone 86-01-69156446
Organization name Peking Union Medical College & Chinese Academy of Medical Science
Department Dept. Biochem & Mol Biol
Lab 621
Street address Dongdansantiao 5hao
City Beijing
ZIP/Postal code 10005
Country China
Platform ID GPL21273
Series (1)
GSE93924 Heterochromatin Protein 1γ Regulates Epigenetic Reprogramming in Primordial Germ Cells
BioSample SAMN06250165
SRA SRX2510725

Supplementary file Size Download File type/resource 368.4 Mb (ftp)(http) BW
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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