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Sample GSM4958888 Query DataSets for GSM4958888
Status Public on Jun 27, 2021
Title BAP1-KO-plus-EV-HiC
Sample type SRA
 
Source name mESC
Organism Mus musculus
Characteristics sample type: BAP1-KO-plus-EV-HiC
Treatment protocol Indicated cells were treated for 48 hours with 500nM dTAG-13 (TOCRIS), or DMSO as vehicle, in order to degrade dTAG-BAP1. To generate stable KO cell lines, 10ug pX458 2.0 plasmid pairs (Addgene) encoding Cas9 and sgRNAs were transfected using Lipofectamine 2000 (Invitrogen), according to manufacturer’s instruction. Sorting of GFP positive cells was carried out 48 hours after transfection and 1000 cells were seeded onto a 15-cm dish. Clones were isolated 10-14 days later and grown further before screening for knockout by Western blot. For rescue clone generation, mESCs were transfected with 10ug pCAG vectors encoding 2xFlag-HA-tagged BAP1 wild-type or BAP1 C91S using Lipofectamine 2000 (ThermoFisher Scientific), according to manufacturer’s instructions. 24 hours post-transfection puromycin selection (1µg/ml) was added for a further 24 hours. Cells were then split to clonal density (~1:40) onto a 15cm plate. Clones were isolated 10-14 days later and grown further before screening for rescue allele expression by Western blot.
Growth protocol mESCs were grown on 0.1% gelatin-coated dishes in 2i/LIF-containing GMEM medium (Euroclone) supplemented with 20% fetal calf serum (Euroclone), 2 mM glutamine (Gibco), 100 U/ml penicillin, 0.1 mg/ml streptomycin (Gibco), 0.1 mM non-essential amino acids (Gibco), 1 mM sodium pyruvate (Gibco), 50 µM ß-mercaptoethanol phosphate buffered saline (PBS; Gibco), 1000 U/ml leukemia inhibitory factor (LIF; produced in-house), and GSK3β and MEK 1/2 inhibitors (ABCR GmbH) to a final concentration of 3 mM and 1 mM, respectively.
Extracted molecule genomic DNA
Extraction protocol Genomic DNA extraction for the HiC was performed as described in Rao et al., 2014.
Library preparation was performed as described in Mumbach et al., 2016.
 
Library strategy Hi-C
Library source genomic
Library selection other
Instrument model Illumina NovaSeq 6000
 
Data processing Alignment - detection of pairs - filtering of valid pairs; HiCpro v2.11.4
mm10
.allValidPairs; interaction matrices generated by the HiCpro pipeline
 
Submission date Dec 06, 2020
Last update date Jun 27, 2021
Contact name Federico Rossi
E-mail(s) federico.rossi@ieo.it
Organization name Istituto Europeo Oncologia
Street address Via Adamello 16
City Milan
ZIP/Postal code 10142
Country Italy
 
Platform ID GPL24247
Series (1)
GSE162739 BAP1 activity regulates Polycomb occupancy and global chromatin condensation counteracting diffuse PCGF3/5-dependent H2AK119ub1 deposition
Relations
BioSample SAMN17011928
SRA SRX9637882

Supplementary file Size Download File type/resource
GSM4958888_BAP1-KO-plus-EV-HiC.allValidPairs.txt.gz 2.6 Gb (ftp)(http) TXT
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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