Nature 2020 01 30 Part.02

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nature research | reporting summary

October 2018

ChIP-seq

Data deposition

Confirm that both raw and final processed data have been deposited in a public database such as GEO.

Confirm that you have deposited or provided access to graph files (e.g. BED files) for the called peaks.

Data access links

May remain private before publication.

NCBI GEO accession ID: GSE114444 (This study) Contains H3 ChIP-seq, Hmo1tag protein ChIP-seq, ChIA-PET RAW &

processed data

Files in database submission GSM3664900 ChIP-seq: WT-Controlplasmid-nucleosome-Input

GSM3664901 ChIP-seq: WT-Controlplasmid-nucleosome-IP

GSM3664902 ChIP-seq: Top2-1-Top1-Controlplasmid-nucleosome-Input

GSM3664903 ChIP-seq: Top2-1-Top1-Controlplasmid-nucleosome-IP

GSM3664904 ChIP-seq: WT-TopAplasmid-nucleosome-Input

GSM3664905 ChIP-seq: WT-TopAplasmid-nucleosome-IP

GSM3664906 ChIP-seq: Top2-1-Top1-TopAplasmid-nucleosome-Input

GSM3664907 ChIP-seq: Top2-1-Top1-TopAplasmid-nucleosome-IP

GSM3664908 ChIP-seq: Hmo1tag-Top2-1-Top1-Controlplasmid-protein-Input

GSM3664909 ChIP-seq: Hmo1tag-Top2-1-Top1-Controlplasmid-protein-IP

GSM3664910 ChIP-seq: Hmo1tag-Top2-1-Top1-TopAplasmid-protein-Input

GSM3664911 ChIP-seq: Hmo1tag-Top2-1-Top1-TopAplasmid-protein-IP

GSM4094803 ChIA-PET: Top2-ChIA-PET

Genome browser session

(e.g. UCSC)

(i) H3 ChIP-seq (Nucleosome):

http://epigenomegateway.wustl.edu/legacy/?genome=sacCer3&session=2IU7XHKkwi&statusId=1279418941

(Note: Use the Nucleosome_TopA Session: First and second track is WT-control plasmid (bed density and bedgraph), third

and fourth track is WT-TopA plasmid (bed density and bedgraph), fifth and sixth track is Top2-1-Top1-Control plasmid (bed

density and bedgraph), seventh and eighth track is Top2-1-Top1-TopA plasmid (bed density and bedgraph)

(ii) Protein ChIP-seq:

http://epigenomegateway.wustl.edu/legacy/?genome=sacCer3&session=4FQoSLC1lk&statusId=1043438118

(Note: Use the TopA_Hmo1 Session: First track is Hmo1tag-Top2-1-Top1-Controlplasmid and Second track is Hmo1tag-

Top2-1-Top1-TopAplasmid)

(iii) ChIA-PET:

http://epigenomegateway.wustl.edu/legacy/?genome=sacCer3&session=arHkgZiCZP&statusId=302725081

(Note: Use the ChIA-PET-Data Session: First track is Hmo1 protein binding regions, Second track is Top2 protein binding

regions, Third track is ChIA-PET data for Top2-10X Flag and Fourth track is the interaction points for ChIA-PET Top2-10X Flag)

No longer applicable for final submission.

Methodology

Replicates One biological replicate for each sample is generated and analyzed for consistency. (Replicates were not yet deposited in the

GEO)

Sequencing depth (i) H3 ChIP-seq (Nucleosome): Average Reads: ~15 Million Reads, Average Read length: ~180bp, Average Mapped Reads: ~8

Million Reads and single end reads (Ion Torrent Platform)

(ii) Protein ChIP-seq: Average Reads: ~15 Million Reads, Average Read length: ~180bp, Average Mapped Reads: ~8 Million

Reads and single end reads (Ion Torrent Platform)

Antibodies (i) H3 ChIP-seq (Nucleosome): anti-Hstone H3 antibody (Abcam cat no 1791)

(ii) Protein ChIP-seq: anti-Flag (M2-antiflag) Antibody (Sigma-Aldrich cat no F3165)

(iii) ChIA-PET: anti-Flag (M2-antiflag) Antibody (Sigma-Aldrich cat no F3165)

Peak calling parameters (i) H3 ChIP-seq: The raw reads were filtered based on the quality value (-q 20 and –p 30) using FASTX Toolkit. The filtered

reads were aligned to the reference genome (SacCer 2011) using TMAP aligner. The PCR duplicates were removed from the

aligned BAM files using PICARD tools. The BAM files were sorted and indexed for the peak calling using SAMTOOLS. The

bedgraph files were generated by comparing bam files of IP and Input (IP read coverage/Input read coverage) result in the

ratio for every base across the whole genome using DEEPTOOLS (bamCompare) (Ramirez et al., 2014). Finally, peak calling

was performed using DANPOS (dpos) toolkit (Chen et al., 2013) with the IP/Input threshold 1.4 (-q 1.4) where the output

peaks corresponds to the individual nucleosome. The DANPOS was preferred over MACS toolkit for the dynamic nucleosome

analysis at single nucleotide resolution.

(ii) Protein ChIP-seq: The raw reads were filtered based on the quality value (-q 20 and –p 30) using FASTX Toolkit. The

filtered reads were aligned to the reference genome (SacCer 2011) using TMAP aligner. The PCR duplicates were removed

from the aligned BAM files using PICARD tools. Finally, MACS2 tool is used for peak calling with the following parameters (-f

BAM --gsize=1.21e+7 -n ctrl-A -B -p 0.01 --nomodel --extsize 200 --broad).

Data quality The peak fold enrichment were in the range of 1.5 to 3.0