GEO help: Mouse over screen elements for information.
|Public on May 13, 2021
|SA2 on A673 STAG2 KO [BA_A673_STAG2KO_SA2]
|A673 clones with STAG2 knocked-out
|cell line: A673
cell type: Ewing sarcoma cell line
genotype/variation: STAG2 knocked-out
chip antibody: Rabbit anti-STAG2 (ChIP-seq) Cell Signaling Cat#5882 RRID AB_10834529
|Cells (20 million per ChIP reaction for all but EWS/FLI1, which required 40 million cells) were crosslinked with warm 1% methanol-free formaldehyde (ThermoFisher) for 10 minutes at room temperature rotating at 12 RPM. The reaction was quenched by adding glycine to a final concentration of 0.125M and incubating for an additional 5 minutes at room temperature rotating at 12 RPM. Cell pellets were washed three times with ice cold PBS and resuspended in 1 ml of SDS lysis buffer (1% SDS, 10 mM EDTA, 50 mM Tris-HCl pH8) supplemented with protease inhibitor cocktail including phenylmethylsulfonyl fluoride (PMSF) and incubated at RT for 2 minutes with gentle rotation. Lysates were centrifuges at 15,000 G for 10 minutes at 4°C and the pellet was re-suspended in 900 µl of ChIP IP buffer (2:1 SDS lysis buffer : triton dilution buffer), transferred to milliTUBE (covaris). Sonication was performed on a E220 Focus Ultra Sonicator (Covaris) using the setting (duty cycle 5%, peak power 140W, cycles per burst 200, Temperature 4°C, time 30 minutes/millitube). ChIP inputs from sheared chromatin were de-crosslinked by adding de-crosslinking buffer (NaHCO3, NaCl, RNase A, Proteinase K) and incubating for two hours at 65°C in a thermal cycler. The remaining sheared chromatin was incubated with primary antibody coupled to Protein A DynaBead (Beckman Coulter, antibody bead conjugation was performed for 16 hours) overnight, rotating at 4°C. As a calibration control, antibody against a drosophila specific histone variant H2Av and drosophila chromatin (active motif) were used as per the recommendation of the manufacturer. The next day, ChIP product was eluted from the Dynabeads in 100 µl of elusion buffer and de-crosslinked for 12 hours at 65°C. For both Input and chipped material, AMPure XP beads (Beckman coulter) were used to purify DNA.
Chromatin immunoprecipitation sequencing for EWS/FLI1, SMC1A, STAG1, STAG2, H3K27me3: ChIP-seq libraries were prepared using Swift S2 Acel reagents on a Beckman Coulter Biomek i7 liquid handling platform from approximately 1 ng of DNA according to the manufacturer’s protocol and 14 cycles of PCR amplification. Finished sequencing libraries were quantified by a Qubit fluorometer and samples were QC’D using a Bioanalyzer Tapestation (Agilent Technologies 2200) to determine fragment size. Library pooling and indexing was evaluated with shallow sequencing on an Illumina MiSeq. Subsequently, libraries were sequenced on a NovaSeq targeting 40 million 100bp read pairs by the Molecular Biology Core facilities at Dana-Farber Cancer Institute. Chromatin immunoprecipitation sequencing for H3K27ac: Chromatin was sheared to about 200 bp fragments using the Covaris ultra-sonication. The lysate was diluted with ChIP dilution buffer (16.7 mM Tris-HCl pH 8.1, 16.7 mM NaCl, 1.2 mM EDTA, 1.1% Triton-X100, 0.01% SDS) and cleared by centrifugation. 5% of the supernatant volume set aside as an input control. The remaining lysate was incubated over night at 4°C with 5 ug of antibody (3 ug for histone antibodies).
|Illumina NovaSeq 6000
|Quality control tests for unmapped sequences were performed based on the FastQC v.0.11.5 software (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/). All of the ChIP-Seq data sets were aligned to the GRCh37/hg19 human genes and Drosophila Melanogaster dm6 genes using bowtie2-2.3.5 with the standard options. PCR duplicates were removed with the Picard v2.18.2 MarkDuplicates tool (Li and Durbin, 2010). The Active Motif Spike-in Normalization protocol was then applied to each hg19 sample by multiplying the human tag counts with the normalization factors derived from the uniquely mapped Drosophila reads as ratios between the sample with the lowest dm6 counts vs. the dm6 counts for that sample. For each mark the reads mapped on the STAG2 wild-type clones A673.sgNT-1c4 and A673.sgNT-2c3 were merged and labeled as "STAG2 WT" and similarly, the reads mapped on the STAG2 knockout clones A673.sgSTAG2-1c6, A673.sgSTAG2-4c5 were merged and labeled as "STAG2 KO". The mapped reads for individual and merged clones were normalized in units of Reads Per Kilobase per Million (RPKM or rpm/bp) and coverage tracks for the RPKM signal were created as bigwig files for bins of size 20 base pairs by using the bamCoverage tool available in deepTools v2.5.3. (Ramirez et al., 2016).
Peak calling was performed against input control using the model-based MACS2 v188.8.131.5260309 software (Zhang et al., 2008), with the cut-off FDR ≤ 0.01. Narrow peaks were identified for all marks except H3K27Me3 for which macs2 was called with the broad peak option. Area under Curve (AUC) RPKM normalized signal across genomic regions was computed with the bwtool software (Pohl and Beato, 2014). Peaks with low area under curve coverage (< 300 RPKM) were disregarded and the ENCODE black-listed regions for hg19 (available at https://www.encodeproject.org/annotations/ENCSR636HFF/) were removed from each set of peak regions. Quality control tests for the mapped reads were performed by using the ChIPQC library available from Bioconductor v3.6 (Carroll et al., 2014). The distances between replicates for STAG2 WT and STAG2 KO clones were estimated based on the multiBamSummary function available from the deepTools v2.5.3. (Ramirez et al., 2016) and visualized on correlation heatmaps and PCA plots. The peaks were annotated with the closest hg19 genes by using the annotatePeaks function available in the Homer v4.11 package (Heinz et al., 2010) and the GREAT annotation platform (McLean et al., 2010).
Supplementary_files_format_and_content: Bigwig files for normalized coverage signal in units of reads per million mapped reads per bp (rpm/bp). Coverage was computed for bins of size 20 base pairs by using the bamCoverage module available in deepTools v2.5.3. The Active Motif Spike-in Normalization protocol was then applied to each hg19 sample -except Input- by multiplying the human tag counts with the normalization factors derived from the uniquely mapped Drosophila reads as ratios between the sample with the lowest dm6 counts vs. the dm6 counts for that sample.
|Jan 29, 2021
|Last update date
|May 13, 2021
|Computational Biology and Bioinformatics
|415 Main St.
|The effect of STAG2 loss in Ewing sarcoma
|The effect of STAG2 loss in Ewing sarcoma [ChIP-seq]