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Sample GSM5315884 Query DataSets for GSM5315884
Status Public on Jun 27, 2021
Title PclBap1KO-H2AK119Ub1
Sample type SRA
Source name Mouse Embryonic Stem Cells
Organism Mus musculus
Characteristics cell type: Mouse embryonic stem cells
chip antibody: H2AK119Ub1
sample type: PclBap1KO-H2AK119Ub1
Treatment protocol To generate stable KO cell lines, 10ug pX458 2.0 plasmid pairs (Addgene) encoding Cas9 and sgRNAs (Table S3) 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 (both E14 and TCF background) 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 μM and 1 μM, respectively. For ATRA stimulation of ESCs, cells were washed twice with PBS 24 hours after seeding and media was replaced with ESC media lacking LIF, GSK3β and MEK1/2 inhibitors, and instead supplemented with 1μM ATRA for 24 hours.
Extracted molecule genomic DNA
Extraction protocol ChIP-seq: DNA was purified with QIAquick PCR purification kit (Qiagen) according to manufacturer’s instructions.
DNA was purified with QIAquick PCR purification kit (Qiagen) according to manufacturer’s instructions. 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 and sequenced on an Illumina HiSeq 2000.
RNA-seq: RNA-seq was performed following SMART-seq2 protocol (Picelli et al., 2014)  with minor modifications. Briefly, poly-A containing mRNA molecules obtained from 1 μg of total RNA were copied into first-strand cDNA by reverse transcription and template-switching using oligo (dT) primers and an LNA-containing template-switching oligo (TSO). Resulting cDNA was pre-amplified with KAPA HotStart Taq enzyme (Kapa Biosystems) and then purified with Ampure beads (Agencourt AMPure XP- Beckman Coulter). One nanogram of pre-amplified cDNA was tagmented with in-house produced Tn5 transposase and further amplified with KAPA HotStart Taq enzyme. After purification with Ampure beads, the quality of the obtained library was assessed by Bioanalyzer (High Sensitivity DNA kit, Agilent Technologies), prior to sequencing. Each RNA-seq experiment was performed in triplicate.
Hi-C: In situ Hi-C was performed as described (Rao et al., 2014) with slight alterations to the protocol. 5x10^6 ESCs were crosslinked for 10 minutes in 1% formaldehyde before quenching in 125mM Glycine for 5 minutes. Cell pellets were washed twice in PBS before contact generation. Pellet was resuspended in 500uL Hi-C lysis buffer (10mM Tris-HCl pH8.0, 10mM NaCl, 0.2% NP40 and protease inhibitors) and incubated with rotation for 30 minutes at 4°C. Cells were pelleted and washed in 500uL Hi-C lysis buffer. Pellet was resuspended in 100uL 0.5% SDS and incubated for 10 minutes at 62°C. 285uL of H20 and 50uL of 10% Triton X-100 was added to quench the SDS at 37°C for 15 minutes. 50uL of 10xNEB DpnII buffer and 750U of DpnII were added (NEB R0453M) then incubated at 37°C overnight with shaking. Digestion was heat inactivated the next day for 20 minutes at 62°C. Biotin fill in was performed by addition of 52uL fill-in mix (0.288mM dCTP/dTTP/dGTP, 50U DNA Polymerase I, Large Klenow fragment (NEB M0210) and 0.288mM biotin-dATP (Thermo 19524016)). Fill-in reaction was incubated at 37°C for 1 hour with shaking. Ligation was performed by adding 150uL 10x NEB T4 DNA ligase buffer, 125uL 10% Triton X-100, 3uL 50mg/mL BSA and 8000U of T4 DNA ligase and H20 up to 1.5mL. Ligation was performed overnight at 25°C with shaking. Nuclei were pelleted before resuspending in IP buffer (33 mM Tris-HCl pH 8, 100 mM NaCl, 5 mM EDTA, 0.2% NaN3, 0.33% SDS, 1.66% Triton X-100) and sonicating to fragment size between 200-1000bp. 15uL of the sample was de-crosslinked overnight at 65°C with 85ul of decrosslinking buffer (0.1M NaHCO3, 1% SDS) before clean up using QiaQuick PCR clean up kit from Qiagen.
Library prep was performed as described (Mumbach et al., 2016). Streptavidin C-1 beads (Thermo 65001) were washed and resuspended in 2x biotin binding buffer (10mM Tris-HCl pH7.5, 1mM EDTA, 2M NaCl). 10ng of DNA was used for biotin capture, streptavidin C-1 beads were added and incubated for 15 minutes at room temperature with shaking. Beads were washed twice at 55°C for 2 minutes in Tween wash buffer (5mM Tris-HCl pH7.5, 0.5mM EDTA, 1M NaCl, 0.05% Tween). Beads were washed once in 1xTD buffer (20mM Tris-HCl pH7.5, 10mM MgCl2, 20% DMF) before being tagmented with in-house produced Tn5 transposase in TD buffer for 55°C for 10 minutes. TN5 was quenched in 50mM EDTA for 30 minutes at 55°C before washing twice in 50mM EDTA, twice in Tween wash buffer and once in 10mM Tris. PCR amplification was carried out on beads using KAPA HotStart Taq enzyme. After purification of PCR product with Ampure beads, the quality of the obtained library was assessed by Bioanalyzer (High Sensitivity DNA kit, Agilent Technologies), prior to sequencing.
Library strategy ChIP-Seq
Library source genomic
Library selection ChIP
Instrument model Illumina NovaSeq 6000
Data processing For ChIP-seq data, paired-end DNA reads were processed through fastp to trim adapters and to remove low quality nucleotides at read ends (Chen et al., 2018). Quality-filtered DNA reads were aligned to the mouse reference genome mm10, or mm10 and fly reference genome (dm6) for histone ChIP-Rx using Bowtie v1.2.2 retaining only uniquely aligned reads (-m 1) and using the parameters -I 10, -X 1000 (Langmead et al., 2009). Reads mapped to both mm10 and dm6 were discarded. Peaks were identified using MACS2 v2.1.1 in narrow mode with parameters –format BAMPE –keep-dup all -m 3 30 and p-value 1e-10 (Zhang et al., 2008). Peaks were annotated using the R package ChIPpeakAnno v3.15 using for each peak the 5 kbp region around the center of the peak  (Zhu et al., 2010).
For RNA-seq, reads were aligned to the mouse reference genome mm10 using STAR v2.7 without allowing for multimapping. PCR duplicates were removed using samblaster (Faust and Hall, 2014). Mapped reads were assigned to genes using featureCounts (Liao et al., 2014) with unstranded read counting on exons and using the gene name as attribute type in the annotation. Genes were annotated as in Gencode M21 (GRCm38) downloaded from Differentially expressed genes were identified using the R package DESeq2 v1.24 using default parameters (Love et al., 2014). The fold change of lowly expressed genes was corrected using the lfcShrink function of the apeglm R package with the type option set to apeglm v1.6 (Zhu et al., 2019). The adjusted p-value was corrected by the independent hypothesis weighting (IHW) method as implemented in the R package IHW v1.12 (Ignatiadis et al., 2016). Genes were considered differentially expressed when presenting an absolute log2 fold change equal or greater than 1.5 and an adjusted p-value lower than 0.05.
For HiC data, DNA reads were filtered for low-quality bases and adapters using fastp (Chen et al., 2018). Filtered DNA reads were processed through the HiCpro pipeline to obtain contact matrices reporting the chromatin interactions at the genome-wide level (Servant et al., 2015). Using default settings, DNA reads were aligned to the mouse reference genome mm10 using bowtie2 in end-to-end mode, uniquely aligned DNA reads were assigned to the DpnII genomic restriction fragments, valid interactions were identified and used to generate interaction matrices. The HiCpro matrices containing all of the valid pairs were converted in hic format using the hicpro2juicebox function (Robinson et al., 2018), and in cool format using the hic2cool function ( Contact matrices in cool format where corrected using the ICE algorithm as implemented in the hicCorrectMatrix function (Wolff et al., 2020). Plots of the interactions occurring at single locus or whole-chromosome level were obtained by HiCplotter (Akdemir and Chin, 2015). Hi-C was performed in duplicate with key findings being overserved in each before pooling replicates to make the final high-depth analyses.
Genome_build: mm10 (GRCm38)
Supplementary_files_format_and_content: bed (MACS2), counts (counts.tsv), differential expression (DESeq2), validpairs (HiCPro).
Submission date May 14, 2021
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 SAMN19217306
SRA SRX10895584

Supplementary file Size Download File type/resource
GSM5315884_PclBap1KO-H2AK119Ub1_E14-input_peaks_p10.bed.gz 120.1 Kb (ftp)(http) BED
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Raw data are available in SRA
Processed data provided as supplementary file

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