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Sample GSM4809406 Query DataSets for GSM4809406
Status Public on Sep 29, 2020
Title v6.5_Flag_Hmga2_input
Sample type SRA
Source name v6.5_Flag_Hmga2_input
Organism Mus musculus
Characteristics cell type: embryonic stem cell
culture condition: 2i
Treatment protocol Beta-catenin knowndown in ES cell and epiLC
Growth protocol 2i media
Extracted molecule genomic DNA
Extraction protocol A total of 2×10^7 ES cells were used per ChIP assay. Cells were washed with 1 x PBS and crosslinked with 1% paraformaldehyde for 10 min at RT and quenched with 1ml 2.5M glycine. Cells were washed with cold PBS three times and resuspended in 300μl lysis buffer. The cell suspension was sonicated in Bioruptor Pico sonication device with the setting of 30 sec on, 30 sec off for 16 cycles followed with centrifuga- tion at maximum speed for 10 min. The supernatant was saved and mixed with antibody coated protein G dynabeads(Invitrogen 10003D) at a ratio of 5μl per 1μg antibody for immunoprecip- itation overnight at 4°C . The next day the protein G dynabeads were washed with 800μl RIPA buffer three times. After the elution, 4μ l RNase A(10μ g/ml) was then added with the beads being incubated at 37°C for 2 hours, after which 2μl Protease K(Invitrogen 20mg/ml) was added and the beads were incubated at 55 °C for 2 hours. After the RNase A and Protease K treatment, the beads were incubated at 55 °C overnight for decrosslinking. The next day the DNA on beads was extracted by 300 μl phenol chloroform isoamyl alcohol(25:24:1) with centrifugation at 12,000 rpm for 5 min at RT. The supernatant was transferred to a new 1.5ml tube with 12μl of 5M NaCl and 2μl glycogen (20mg/ml). The DNA was precipitated by 750μl cold 100% ethanol with at least 30 min incubation at - 80 °C . The sample was centrifuged for 30 min in 4 °C at maximum speed and the ethanol was decanted. The pellet was washed with 800 μ l cold 70% ethanol, centrifuged and air dried. The DNA was resuspended in 55μl nuclease-free water.
RNA-seq and ChIP-seq libraries were prepared using the TruSeq RNA kit for sequencing,
Library strategy ChIP-Seq
Library source genomic
Library selection ChIP
Instrument model Illumina HiSeq 2500
Description Sonicated whole cell extract
Data processing Basecalling and bcl to fastq generation was done using bcl2fastq2 v2.18 from illumina.
RNA-seq was aligned to genome mm10 from UCSC using STAR aligner
TPM expression was generated using RSEM
ChIP-seq was aligned to genome mm10 using bowtie2
Bigwig files are normalized and extended to fragment length for IP samples
Genome_build: mm10
Supplementary_files_format_and_content: RNA-seq TPM in excel file, ChIP-seq in bigwig files
Submission date Sep 29, 2020
Last update date Oct 01, 2020
Contact name Shiyuan (Cynthia) Chen
Organization name Stowers Institute for Medical Research
Department Computational Biology
Street address 1000 E 50th St
City Kansas City
State/province MO
ZIP/Postal code 64110
Country USA
Platform ID GPL17021
Series (1)
GSE108620 β-catenin associated protein complex maintains ground state mouse embryonic stem cell by regulating lineage development
BioSample SAMN16289279
SRA SRX9213051

Supplementary file Size Download File type/resource 315.9 Mb (ftp)(http) BW
SRA Run SelectorHelp
Processed data provided as supplementary file
Raw data are available in SRA

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