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Sample GSM4195088 Query DataSets for GSM4195088
Status Public on Dec 01, 2019
Title SAFB-K9me3-HiChip-Rep2
Sample type SRA
Source name AML12 shSafb
Organism Mus musculus
Characteristics cell line: AML12
lentivirus: shSafb
antibody: H3K9me3
Treatment protocol The shRNA lentiviruses were prepared as previously described (Fu et al., 2015). Then AML12 cells were infected with 200 μl of virus concentrate in a well of six-well-plates. Medium was changed 24 hours after transfection, and then collect cells 5 days after transfection.
Growth protocol AML12 cells were cultured in DMEM/F12 supplemented with 10% fetal bovine serum, ITS Liquid Media Supplement, and 0.1μM dexamethasone at 37 °C and 5% CO2.
Extracted molecule genomic DNA
Extraction protocol The HiChIP of H3K9me3 was performed as described (Mumbach et al., 2016), started with 2.5 millions of AML12 cells for each sam- ple. Briefly, 2.5 millions of cells were digested by 100U of MboI overnight at 37 C. After biotin filling and DNA ligating, the nuclear were resuspended by sonication buffer (1%SDS, 10mM EDTA, 50mM Tris-HCl, 20mM NaBu, 1 3 Proteinase Inhibitor), and chromatin was sheared into 300-700bp using Bioruptor followed by incubating with antibody overnight at 4 C. 25 mL beads were washed once with cold 0.5% BSA, and added into the sheared chromatin-protein supernatant for 2 hours at 4 C. Washed with high salt wash buffer (50mM HEPEs, 500mM NaCl, 1mM EDTA, 0.1%DOC, 1%Triton X-100) three times; low wash buffer (10mM Tris-HCl, 250mM LiCl, 1mM EDTA, 0.5%NP40, 0.5%DOC) twice; TE once. Chromatin-protein complex were eluted for 15min at 65 C in 50 mL elution buffer (1% SDS in TE). After reverse-crosslinking, DNA was extracted using ultrapure phenol/chloroform. Chromatin fragments con- taining biotin were captured by MyOne Streptatvidin beads (ThermoFished 65601), and libraries were prepared on beads using Tn5 from DNA Library Prep Kit V2 (Vazyme TD503) for 30min at 37 C, followed by amplification.
Illumina sequencing library were prepared according to the standard Illumina protocol.
Library strategy OTHER
Library source genomic
Library selection other
Instrument model HiSeq X Ten
Data processing Library strategy: HiChIP
Hi-Chip paired-end reads were aligned to mm9 reference genome using the HiC-Pro pipeline (Servant et al., 2015) with default set- tings. We converted .all ValidPairs files to .hic files by the script from HiC-Pro utilities. To detect the differences between experiments, we normalized the interaction matrices for each chromosome separately according to the total contacts in each chromosome. This normalization led to the sum of contacts in each chromosome to 1e8.
Genome_build: mm9
Submission date Nov 27, 2019
Last update date Dec 01, 2019
Contact name Bo Wen
Organization name Fudan University
Street address 130 Dongan Rd.
City ShangHai
ZIP/Postal code 200032
Country China
Platform ID GPL21273
Series (2)
GSE125037 The Nuclear Matrix Protein SAFB Cooperates with Major Satellite RNAs to Stabilize Heterochromatin Architecture Partially through Phase Separation
GSE141113 The nuclear matrix protein SAFB maintains heterochromatin architecture through RNA-dependent phase separation [HiChIP]
BioSample SAMN13412359
SRA SRX7228450

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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