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Sample GSM4958803 Query DataSets for GSM4958803
Status Public on Jun 27, 2021
Title BAP1-KO-clone1-input.2
Sample type SRA
Source name mESC
Organism Mus musculus
Characteristics chip antibody: Input
sample type: BAP1-KO-clone1-input
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 ChIP experiments were performed according to standard protocols as described previously (Ferrari et al., 2014)
DNA libraries were prepared with 2–10 ng of DNA using an in-house protocol (Blecher-Gonen et al., 2013) by the IEO genomic facility
Library strategy ChIP-Seq
Library source genomic
Library selection ChIP
Instrument model Illumina NovaSeq 6000
Description Low-depth sequenced genomic DNA input for the normalisation of BAP1-KO-clone1-H3K27me2, BAP1-KO-clone1-H3K27ac, BAP1-KO-clone1-H3K4me3, BAP1-KO-clone1-H3K4me1
Data processing Alignment; bowtie v1.2.2; --chunkmbs 1024 -m 1 --best -S --no-unal -q -I 10 -X 1000
Peak calling; MACS2 v2.1.1 narrow mode; –format BAMPE –keep-dup all -m 3 30 pvalue 1e-10
bed file format; narrow peaks called with MACS2 having a -log10 p-value higher than 10
Submission date Dec 06, 2020
Last update date Jun 27, 2021
Contact name Federico Rossi
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
BioSample SAMN17011957
SRA SRX9637885

Supplementary data files not provided
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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