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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
mm10
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

E-mail(s)

federico.rossi@ieo.it

Organization name

Istituto Europeo Oncologia

Street address

Via Adamello 16