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ChIP-seq Analysis of Histone H3K27ac and H3K27me3 Showing Different Distribution Patterns in Chromatin
Biomed Sci Letters 2022;28:109-119
Published online June 30, 2022;  https://doi.org/10.15616/BSL.2022.28.2.109
© 2022 The Korean Society For Biomedical Laboratory Sciences.

Jin Kang* and AeRi Kim†,* *

Department of Molecular Biology, College of Natural Sciences, Pusan National University, Busan 46241, Korea
Correspondence to: AeRi Kim. Department of Molecular Biology, College of Natural Sciences, Pusan National University, Busan 46241, Korea.
Tel: +82-51-510-3684, Fax: +82-51-513-9258, e-mail: kimaeri@pusan.ac.kr
*Post-Doctor, **Professor.
Received June 3, 2022; Revised June 21, 2022; Accepted June 22, 2022.
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
 Abstract
Histone proteins can be modified by the addition of acetyl group or methyl group to specific amino acids. The modifications have different distribution patterns in chromatin. Recently, histone modifications are studied based on ChIP-seq data, which requires reasonable analysis of sequencing data depending on their distribution patterns. Here we have analyzed histone H3K27ac and H3K27me3 ChIP-seq data and it showed that the H3K27ac is enriched at narrow regions while H3K27me3 distributes broadly. To properly analyze the ChIP-seq data, we called peaks for H3K27ac and H3K27me3 using MACS2 (narrow option and broad option) and SICER methods, and compared propriety of the peaks using signal-to-background ratio. As results, H3K27ac-enriched regions were well identified by both methods while H3K27me3 peaks were properly identified by SICER, which indicates that peak calling method is more critical for histone modifications distributed broadly. When ChIP-seq data were compared in different sequencing depth (15, 30, 60, 120 M), high sequencing depth caused high false-positive rate in H3K27ac peak calling, but it reflected more properly the broad distribution pattern of H3K27me3. These results suggest that sequencing depth affects peak calling from ChIP-seq data and high sequencing depth is required for H3K27me3. Taken together, peak calling tool and sequencing depth should be chosen depending on the distribution pattern of histone modification in ChIP-seq analysis.
Keywords : H3K27ac, H3K27me3, ChIP-Seq, Peak calling, Sequencing depth
꽌 濡

엳뒪넠 吏꾪빑꽭룷쓽 빑 궡뿉꽌 DNA 寃고빀븯뿬 겕濡쒕쭏떞(chromatin)쓣 援ъ꽦븯뒗 援ъ“ 떒諛깆쭏씠떎(Wolffe and Guschin, 2000; Cutter and Hayes, 2015). 4醫낅쪟쓽 엳뒪넠 떒諛깆쭏(H2A, H2B, H3, H4) 2媛쒖뵫 寃고빀븯뿬 엳뒪넠 뙏웾泥(histone octamer)瑜 씠猷⑤ʼn, 씠 뙏웾泥대뒗 빟 146 bp쓽 DNA뿉 媛먭꺼 겕濡쒕쭏떞쓽 援ъ“쟻 湲곕낯 떒쐞泥댁씤 돱겢젅삤醫(nucleosome)쓣 삎꽦븳떎. 씠 븣, 엳뒪넠怨 DNA 궗씠쓽 젙쟾湲곗쟻씤 씤젰씠 돱겢젅삤醫쓣 삎꽦븯뒗 湲곕낯 썝由ъ씠硫, 씠瑜 諛뷀깢쑝濡 湲 DNA 媛떏쓣 泥닿퀎쟻쑝濡 쓳異뺥븯뿬 겕濡쒕쭏떞 援ъ“瑜 留뚮뱺떎. 븯吏留 쟾궗씤옄(transcription factor)굹 RNA 以묓빀슚냼(polymerase) 엯옣뿉꽌 蹂대㈃ 돱겢젅삤醫 씠 떒諛깆쭏뱾씠 DNA뿉 寃고빀븯뒗 寃껋쓣 留됰뒗 옣븷臾쇱씠떎. 씠瑜 빐寃고븯湲 쐞빐 DNA 엳뒪넠 궗씠쓽 寃고빀쓣 빟솕떆궎뒗 諛⑸쾿씠 議댁옱븯뒗뜲, 엳뒪넠 떒諛깆쭏쓽 솕븰쟻 蹂삎씠 以묒슂븳 遺遺꾩쓣 떞떦븳떎.

엳뒪넠 떒諛깆쭏쓽 N-留먮떒 븘誘몃끂궛뿉뒗 븘꽭떥湲곕굹 硫뷀떥湲 媛숈 솕븰 洹몃9씠 媛뿭쟻쑝濡 寃고빀븷 닔 엳뒗뜲, 씠 솕븰쟻 蹂삎뱾쓣 엳뒪넠 蹂삎(histone modifications)씠씪 씪而ル뒗떎(Bannister and Kouzarides, 2011). 엳뒪넠 蹂삎 븘誘몃끂궛쓽 쐞移섏 寃고빀븯뒗 솕븰 洹몃9쓽 醫낅쪟뿉 뵲씪 떎뼇븯硫, 吏곸젒쟻쑝濡 겕濡쒕쭏떞 援ъ“瑜 蹂솕떆궎嫄곕굹 援ъ“瑜 蹂솕떆궎뒗 떒諛깆쭏쓣 紐⑥쭛븯뒗뜲(recruit) 愿뿬븳떎. 븘꽭떥솕(acetylation) 硫뷀떥솕(methylation)뒗 몴쟻씤 엳뒪넠 蹂삎쑝濡 二쇰줈 由ъ떊(lysine, K) 옍湲곗뿉꽌 씪뼱궃떎. 븘꽭떥솕쓽 寃쎌슦, 엳뒪넠 떒諛깆쭏쓽 뼇쟾븯瑜 媛먯냼떆궎硫, 洹 寃곌낵 DNA쓽 寃고빀쓣 빟솕떆耳 겕濡쒕쭏떞 援ъ“瑜 솢꽦솕떆궓떎. 씠뿉 諛섑빐 硫뷀떥솕뒗 蹂삎씠 씪뼱굹뒗 븘誘몃끂궛쓽 쐞移섏뿉 뵲씪 洹 뿭븷씠 떎瑜대떎. 엳뒪넠 H3쓽 4踰 由ъ떊쓽 3-硫뷀떥솕(H3K4me3)뒗 쟾궗媛 솢諛쒗엳 씪뼱굹뒗 봽濡쒕え꽣(promoter)뿉꽌 쟾궗씤옄쓽 紐⑥쭛쓣 룄二쇱留(Vermeulen et al., 2007; Lauberth et al., 2013), H3K27me3뒗 겕濡쒕쭏떞 援ъ“瑜 遺덊솢꽦솕떆궎뒗 떒諛깆쭏쓣 遺덈윭뱾뿬 쑀쟾옄 쟾궗 뼲젣뿉 湲곗뿬븳떎(Cao et al., 2002; Min et al., 2003). 듅엳 엳뒪넠 H3K27ac H3K27me3뒗 媛숈 븘誘몃끂궛뿉꽌 씪뼱굹吏留 겕濡쒕쭏떞 援ъ“뿉 誘몄튂뒗 쁺뼢씠 긽諛섎릺湲 븣臾몄뿉 씠뱾 蹂삎뿉 븳 留롮 뿰援щ뱾씠 닔뻾릺뼱 솕떎(Kim and Kim, 2011; Bowman et al., 2014; Chrysanthou et al., 2022). 삉븳 엳뒪넠 蹂삎씠 씪뼱굹뒗 쑀쟾泥 긽쓽 湲몄씠, 利 遺꾪룷 삎깭룄 엳뒪넠 蹂삎쓽 醫낅쪟뿉 뵲씪 떎瑜대떎(Landt et al., 2012). 삁瑜 뱾뼱 H3K4me3 H3K27ac뒗 쑀쟾옄쓽 쟾궗떆옉遺쐞(transcription start site) 二쇰쓽 吏㏃(醫곸) 吏뿭뿉꽌 愿李곕릺吏留, H3K9me3굹 H3K27me3뒗 긽쟻쑝濡 湲(꼻) 吏뿭뿉 뿰냽쟻쑝濡 遺꾪룷븯怨 엳떎.

엳뒪넠 蹂삎 븫쓣 룷븿븳 떎뼇븳 吏덈퀝怨 뿰愿꽦쓣 蹂댁씤떎(Chi et al., 2010; Fernandes et al., 2020). 鍮꾩젙긽쟻씤 엳뒪넠 蹂삎 쑀쟾옄쓽 쟾궗 긽깭瑜 蹂솕떆궗 닔 엳쑝硫, 洹 寃곌낵 吏덈퀝쓣 珥덈옒븷 닔 엳떎. 젣1삎 떦눊 솚옄 怨좏삁븬 쑀諛 깮伊 紐⑤뜽뿉꽌 H3K27ac瑜 룷븿븳 엳뒪넠 H3쓽 븘꽭떥솕 닔以씠 鍮꾩젙긽쟻쑝濡 利앷븯쑝硫(Lee et al., 2012; Wang et al., 2019), 쟾由쎌꽑븫, 쐞븫, 痍뚯옣븫뿉꽌뒗 H3K27me3 利앷뿉 쓽븳 븫 뼲젣 쑀쟾옄뱾(tumor suppressor genes)쓽 諛쒗쁽 媛먯냼媛 蹂닿퀬릺뿀떎(Yu et al., 2007; Fujii and Ochiai, 2008; Ougolkov et al., 2008). 씠윭븳 뿰援 寃곌낵瑜 諛뷀깢쑝濡 엳뒪넠 蹂삎 슚냼 쑀쟾옄瑜 몴쟻쑝濡 븳 移섎즺젣媛 媛쒕컻릺怨 엳쑝硫, 떎젣濡 엳뒪넠 H3K27 蹂삎쓣 떞떦븯뒗 enhancer of zeste homolog 2 (EZH2) histone deacetylase (HDAC) 슚냼쓽 뼲젣젣媛 빆븫젣쓽 슚怨쇰 긽듅떆궓떎뒗 蹂닿퀬룄 엳떎(Fiskus et al., 2009). 삉븳 쑀쟾泥 닔以쓽 엳뒪넠 蹂삎 뿰援щ 넻빐 깉濡쒖슫 吏덈퀝 愿젴 쑀쟾옄瑜 룞젙빐궪 닔룄 엳떎. 泥쒖떇, 븣痢좏븯씠癒, 媛묒긽꽑븫 솚옄쓽 꽭룷뿉꽌 닔뻾븳 쑀쟾泥 쟾泥댁쓽 H3K27ac 遺꾪룷 뿰援щ뒗 깉濡쒖슫 吏덈퀝 愿젴 쑀쟾옄瑜 꽑蹂꾪빐깉쑝硫, 移섎즺젣 媛쒕컻쓽 몴쟻 쑀쟾옄 썑蹂대 젣떆븯떎(Marzi et al., 2018; McErlean et al., 2020; Zhang et al., 2021). 씠泥섎읆 엳뒪넠 蹂삎뿉 븳 뿰援 寃곌낵뒗 吏덈퀝쓽 吏꾨떒 諛 삁썑 삁痢, 洹몃━怨 移섎즺젣 媛쒕컻 벑 떎뼇븳 遺꾩빞뿉꽌 솢슜릺怨 엳떎.

엳뒪넠 蹂삎 二쇰줈 硫댁뿭移④컯踰(chromatin immunoprecipitation, ChIP)쓣 씠슜븯뿬 뿰援щ맂떎(Kuo and Allis, 1999). 씠뒗 룷由꾩븣뜲엳뱶濡 怨좎젙맂 겕濡쒕쭏떞쓣 돱겢젅삤醫 떒쐞濡 젅떒븳 썑, 썝븯뒗 엳뒪넠 蹂삎 빆泥댁 諛섏쓳떆궎怨, 移⑥쟾맂 돱겢젅삤醫쓽 DNA瑜 젙젣븯뿬 遺꾩꽍븯뒗 湲곕쾿씠떎. 怨쇨굅뿉뒗 PCR 湲곕쾿쑝濡 ChIPed DNA쓽 듅젙 遺遺꾨쭔쓣 遺꾩꽍븯쑝굹, 理쒓렐뿉뒗 李⑥꽭뿼湲곗꽌뿴遺꾩꽍(next-generation sequencing, NGS)씠씪怨 씪而ル뒗 떆떛 湲곕쾿씠 媛쒕컻릺뼱 쑀쟾泥 닔以쓽 遺꾩꽍씠 媛뒫븯寃 릺뿀떎. 蹂 뿰援щ뒗 遺꾪룷 삎깭媛 떎瑜 엳뒪넠 蹂삎뱾쓽 ChIP-seq 뜲씠꽣瑜 쟻젅엳 遺꾩꽍븯湲 쐞븯뿬, H3K27ac H3K27me3瑜 긽쑝濡 NGS 뜲씠꽣 peak 꽑蹂 봽濡쒓렇옩뱾(MACS2 SICER)쓣 鍮꾧탳븯쑝硫, 씠뱾 諛⑸쾿쓽 쟻젅꽦쓣 peak濡 꽑蹂꾨릺吏 븡 遺遺꾧낵 鍮꾧탳븯뿬 寃利앺븯떎. 삉븳 ChIP-seq 뜲씠꽣瑜 떎뼇븳 슜웾(read 닔)뿉꽌 遺꾩꽍븯뿬 엳뒪넠 蹂삎 遺꾪룷 삎깭뿉 뵲瑜 ChIP-seq 뜲씠꽣쓽 吏덉쟻 닔以怨 떆떛 슜웾(sequencing depth) 궗씠쓽 긽愿愿怨꾨 議곗궗븯떎.

옱猷 諛 諛⑸쾿

Public K562 ChIP-seq 뜲씠꽣 遺꾩꽍

궗엺 쟻삁援 쑀옒 꽭룷二 K562쓽 H3K27ac H3K27me3 ChIP-seq raw fastq 뙆씪 GEO database뿉꽌 솗蹂댄븯쑝硫(H3K27ac, GSE66023; H3K27me3, GSE167869), Galaxy 뵆옯뤌쓣 씠슜븯뿬 遺꾩꽍븯떎(https://usegalaxy.org). 뿼湲곗꽌뿴쓽 젙솗룄媛 궙 reads뒗 Filter by quality FASTQ Quality Trimmer씪뒗 遺꾩꽍 룄援щ 씠슜븯뿬 젣嫄고븯怨, 吏덉쟻 닔以(quality)씠 蹂댁젙맂 reads瑜 Bowtie2瑜 씠슜븯뿬 궗엺 hg19 canonical reference genome뿉 젙젹(alignment, mapping)븯떎(Langmead and Salzberg, 2012). Reads瑜 젙젹븳 bam 뙆씪뿉꽌 mapping quality (MAPQ)媛 20 誘몃쭔씠嫄곕굹 PCR duplicate濡 異붿젙릺뒗 reads瑜 젣嫄고븯뿬 젙솗엳 젙젹맂 reads留뚯쓣 씠썑 遺꾩꽍뿉 궗슜븯떎. ChIP-seq 뜲씠꽣瑜 떆媛곹솕븯湲 쐞빐 BamCoverage瑜 씠슜븯뿬 bigwig 뙆씪쓣 깮꽦븯쑝硫(Ramírez et al., 2016), H3K27ac H3K27me3쓽 遺꾪룷뒗 Integrative genomics viewer (IGV)뿉꽌 愿李고븯떎(Thorvaldsdóttir et al., 2013). UCSC database뿉꽌 젣怨듯븯뒗 궗엺 쑀쟾옄뿉 븳 bed 뙆씪쓣 湲곗쑝濡 쟾궗떆옉遺쐞 二쇰쓽 H3K27ac H3K27me3 遺꾪룷 닔以쓣 deeptools쓽 computeMatrix濡 遺꾩꽍븯怨 plotHeatmap쑝濡 굹궡뿀떎.

엳뒪넠 H3K27 蹂삎쓽 peak 꽑蹂

H3K27ac H3K27me3媛 留롮씠 愿李곕릺뒗 吏뿭(peak)쓣 꽑蹂꾪븯湲 쐞빐 珥 꽭 媛吏 遺꾩꽍 諛⑸쾿쓣 궗슜븯떎. 泥 踰덉㎏濡 MACS2뿉꽌 default parameter濡 몢 蹂삎쓽 narrow peak瑜 꽑蹂꾪븯怨, 룞씪븳 bam 뙆씪뿉꽌 MACS2쓽 broad option쓣 궗슜븯뿬 뜲씠꽣瑜 遺꾩꽍븯떎(Zhang et al., 2008). 留덉留됱쑝濡 SICER뿉꽌 default parameter瑜 씠슜븯뿬 peak瑜 꽑蹂꾪븯怨 鍮꾧탳븯쑝硫(Zang et al., 2009), SICER濡 遺꾩꽍븯湲 쐞빐 bam 뙆씪쓣 bed 뙆씪濡 蹂솚븯뿬 궗슜븯떎. 紐⑤뱺 peak 꽑蹂 怨쇱젙뿉꽌 ChIP 떎뿕뿉 븳 input ChIP-seq 뜲씠꽣瑜 control濡 궗슜븯떎.

Signal-to-background ratio 怨꾩궛

Peak 꽑蹂 諛⑸쾿쓽 쟻젅꽦쓣 뙋떒븯湲 쐞빐 signal-to-background ratio瑜 鍮꾧탳븯떎. 꽭 媛吏 遺꾩꽍 諛⑸쾿뿉 쓽빐 꽑蹂꾨맂 peaks瑜 湲곗쑝濡 엳뒪넠 蹂삎씠 씪뼱굹吏 븡뒗 吏뿭(non-peak regions)쓣 ComplementBed濡 吏젙븯떎. Peaks non-peak regions뿉 젙젹맂 read 닔瑜 痢≪젙븯뿬 counts per million (CPM) 媛믪쓣 怨꾩궛븯쑝硫(CPM=(듅젙 吏뿭쓽 read 닔)/((쟾泥 read 닔)/106)), 씠 븣 쟾泥 read 닔뒗 flagstat쓣 씠슜븯뿬 痢≪젙븯떎. 洹몃━怨 Peaks쓽 룊洹 CPM 媛믪쓣 non-peak regions쓽 룊洹 CPM 媛믪쑝濡 굹닎쑝濡쒖뜥 signal-to-background ratio瑜 룄異쒗븯떎.

꽭룷 諛곗뼇

蹂 떎뿕뿉 궗슜븳 K562 꽭룷二쇰뒗 ATCC뿉꽌 援ъ엯븯쑝硫, 諛곗뒗 Gibco뿉꽌 援ъ엯븳 떆빟뱾濡 젣옉븯떎. 궗슜븳 諛곗뒗 10% FBS, 1% Penicillin/Streptomycin, 2 mM L-Glutamine, 20 mM HEPES媛 룷븿맂 RPMI 1640씠뿀쑝硫, 꽭룷뒗 37℃, 5% CO2 議곌굔뿉꽌 諛곗뼇릺뿀떎.

겕濡쒕쭏떞 硫댁뿭移④컯踰(ChIP)

ChIP 떎뿕 떎쓬怨 媛숈씠 닔뻾븯떎(Kuo and Allis, 1999). K562 (1×107) 꽭룷瑜 1% formaldehyde (Sigma) 議곌굔뿉꽌 10遺꾧컙 泥섎━븳(crosslinking) 썑, 0.125 M Glycine (Invitrogen) 議곌굔쑝濡 諛섏쓳쓣 以묐떒븯떎. Cell lysis buffer瑜 泥섎━븯怨 10遺꾧컙 뼹쓬뿉꽌 諛섏쓳븳 썑, 썝떖遺꾨━瑜 넻빐 빑쓣 遺꾨━븯쑝硫, 遺꾨━븳 빑뿉 MNase (Worthington Biochemical Corp)瑜 37℃, 15遺꾧컙 泥섎━븯뿬 겕濡쒕쭏떞쓣 떒씪 돱겢젅삤醫 닔以쑝濡 젅떒븯떎. 빑쓣 遺꾪빐븳 썑, 젅떒맂 겕濡쒕쭏떞쓣 Protein A agarose beads (Millipore) 諛섏쓳떆耳(4℃, overnight) 硫댁뿭移④컯 쟾뿉 beads뿉 鍮꾪듅씠쟻쑝濡 寃고빀븯뒗 겕濡쒕쭏떞쓣 젣嫄고븯떎(preclearing). 씠썑, 엳뒪넠 H3K27ac 삉뒗 H3K27me3뿉 듅씠쟻씤 빆泥댁 諛섏쓳떆궎怨(4℃, 3떆媛), Protein A agarose beads瑜 씠슜븯뿬 빆泥댁뿉 寃고빀븳 겕濡쒕쭏떞쓣 꽑蹂 遺꾨━븯떎. 걹쑝濡 phenol extraction 湲곕쾿쓣 씠슜븯뿬 DNA瑜 遺꾨━ 젙젣븯떎. ChIP 떎뿕뿉 궗슜븳 빆泥대뒗 Abcam怨 Millipore뿉꽌 援ъ엯븯떎(H3K27ac, ab4729; H3K27me3, 07-449).

ChIP-seq 씪씠釉뚮윭由 젣옉

ChIP-seq 씪씠釉뚮윭由щ뒗 NEBNext Ultra II DNA Library Prep Kit (New England Biolabs)瑜 씠슜븯뿬 젣옉븯쑝硫, H3K27ac H3K27me3뿉 븳 ChIP-seq 씪씠釉뚮윭由 젣옉 蹂 뿰援ъ떎뿉꽌 湲곗〈뿉 닔뻾뻽뜕 議곌굔怨 룞씪븯寃 吏꾪뻾븯떎(Kang et al., 2021a; Kim et al., 2021). ChIPed DNA (H3K27ac, 10 ng; H3K27me3, 1 ng)쓽 뼇 留먮떒쓣 鍮꾩젏李⑹꽦 留먮떒(blunt end)쑝濡 留뚮뱾怨, 3' 留먮떒뿉 븘뜲땶쓣 異붽븳 썑 AT base pairing쑝濡 NEBNext Adapter瑜 뿰寃고븯떎. Adapter媛 뿰寃곕맂 DNA뒗 NEBNext Multiplex Oligos for Illumina (96 Unique Dual Index Primer Pairs)濡 PCR쓣 닔뻾븯뿬 利앺룺떆耳곌퀬 (H3K27ac, 7 cycles; H3K27me3, 11 cycles), NEBNext Sample Purification Beads濡 빟 175 bp 湲몄씠쓽 DNA瑜 꽑蹂꾪븳 썑, 씪씠釉뚮윭由щ 젣옉븯떎. 젣옉븳 씪씠釉뚮윭由ъ쓽 냽룄뒗 Qubit dsDNA HS assay kit (Invitrogen)濡 痢≪젙븯쑝硫, NGS 떆떛 NovaSeq 6000뿉꽌 100 bp paired-end 議곌굔쑝濡 닔뻾븯뿬 빟 6泥쒕쭔媛(60 M)쓽 reads瑜 뼸뿀떎.

ChIP-seq 뜲씠꽣 遺꾩꽍

Paired-end 떆떛쓣 넻빐 뼸 H3K27ac H3K27me3 ChIP-seq 뜲씠꽣 遺꾩꽍 蹂 뿰援ъ떎뿉꽌 湲곗〈뿉 닔뻾븯뜕 遺꾩꽍 怨쇱젙怨 룞씪븯寃 吏꾪뻾븯쑝硫(Kim et al., 2020; Kang et al., 2021b; Kang et al., 2021c), single-end 떆떛쑝濡 뼸 public 뜲씠꽣 遺꾩꽍 諛⑸쾿쓣 씪遺 닔젙븯떎. Fastq 뙆씪쓽 quality뒗 FastQC濡 솗씤븯怨, read 궡 quality媛 궙 뿼湲곕뒗 Trimmomatic쑝濡 젣嫄고븯떎(Bolger et al., 2014). Quality瑜 蹂댁젙븳 reads뒗 hg19 canonical reference genome뿉 Bowtie2濡 젙젹븯怨, MAPQ媛 20 誘몃쭔씠嫄곕굹 PCR duplicate濡 쓽떖릺뒗 reads뒗 젣嫄고븯떎. ChIP-seq 뜲씠꽣쓽 떆媛곹솕瑜 쐞빐 BamCoverage瑜 씠슜븯뿬 bigwig 뙆씪쓣 깮꽦븯怨, IGV瑜 넻빐 蹂삎쓽 遺꾪룷瑜 愿李고븯떎. ChIP-seq 뜲씠꽣쓽 쟾泥 read 닔瑜 젣븳븯湲 쐞빐 Downsample SAM/BAM쓣 씠슜븯뿬 湲곗〈 bam 뙆씪濡쒕꽣 빟 15 M, 30 M, 60 M, 120 M쓽 reads뿉 빐떦븯뒗 bam 뙆씪쓣 깉濡 깮꽦븯쑝硫, 깮꽦맂 bam 뙆씪쓣 bigwig 뙆씪濡 蹂솚븯뿬 媛 엳뒪넠 蹂삎쓽 遺꾪룷瑜 愿李고븯떎. Read 닔瑜 젣븳븳 뜲씠꽣뿉꽌 H3K27ac H3K27me3쓽 peak뒗 MACS2 broad SICER濡 꽑蹂꾪븯怨, 꽑蹂꾪븳 peaks 궗씠뿉 以묒꺽(overlap)릺뒗 peak쓽 媛쒖닔뒗 intervene쑝濡 痢≪젙븯뿬 venn diagram쑝濡 굹궡뿀떎(Khan and Mathelier, 2017).

寃곌낵 諛 怨좎같

엳뒪넠 H3K27 蹂삎쓽 遺꾪룷 삎깭 鍮꾧탳

엳뒪넠 H3K27뿉꽌 씪뼱굹뒗 몢 蹂삎, H3K27ac H3K27me3뒗 겕濡쒕쭏떞뿉꽌 洹 遺꾪룷 삎깭媛 떎瑜 寃껋쑝濡 蹂댁씤떎. 利 H3K27ac뒗 二쇰줈 쟾궗떆옉遺쐞 媛숈 醫곸 遺遺꾩뿉꽌 愿李곕릺吏留, H3K27me3뒗 긽쟻쑝濡 湲 遺遺꾩뿉꽌 뿰냽쟻쑝濡 굹궃떎. 씠윭븳 듅吏뺤쓣 떆媛곹솕븯怨 洹 李⑥씠瑜 鍮꾧탳븯湲 쐞빐 public K562 ChIP-seq 뜲씠꽣瑜 遺꾩꽍븯떎. Fig. 1A뿉꽌 蹂댁뿬吏뒗 寃껋쿂읆, H3K27ac뒗 吏㏃ 湲몄씠뿉 嫄몄퀜 遺꾪룷븯硫, 궡遺뿉 諛吏 遺쐞媛 蹂꾨룄濡 議댁옱븯뒗 뼇긽쓣 蹂댁떎. 븯吏留 H3K27me3뒗 긽쟻쑝濡 湲 遺쐞뿉 洹좎씪븯寃 遺꾪룷븯怨 엳뿀쑝硫, 븯굹쓽 쁺뿭(domain, 룄硫붿씤)쓣 삎꽦븯뒗 寃껋쿂읆 蹂댁떎. 씠윭븳 遺꾪룷 삎깭媛 쑀쟾泥 쟾泥댁뿉꽌 굹굹뒗吏 議곗궗븯湲 쐞븯뿬 쑀쟾옄쓽 쟾궗떆옉遺쐞瑜 湲곗쑝濡 H3K27ac H3K27me3쓽 遺꾪룷 긽깭瑜 鍮꾧탳븯떎(Fig. 1B). H3K27ac 닔以쓣 湲곗쑝濡 쟾궗떆옉遺쐞瑜 굹뿴뻽쓣 븣, H3K27me3뒗 H3K27ac 닔以씠 궙 遺쐞뿉꽌 愿李곕릺뿀떎. 몢 蹂삎쓽 諛고쟻씤 遺꾪룷뒗 쟾궗떆옉遺쐞瑜 H3K27me3 닔以쑝濡 젙젹뻽쓣 븣룄 愿李곕릺뿀떎. 洹몃윭굹 씠뱾 H3K27 蹂삎쓽 遺꾪룷 湲몄씠瑜 鍮꾧탳븯硫 H3K27ac뒗 쟾궗떆옉遺쐞쓽 ±2 Kb 씠궡뿉 二쇰줈 遺꾪룷븯吏留, H3K27me3뒗 ±20 Kb源뚯 꼻 吏뿭뿉 嫄몄퀜 遺꾪룷븯怨 엳뒗 寃껋씠 愿李곕릺뿀떎. 씠뒗 엳뒪넠 H3K27 蹂삎씠 굹굹뒗 湲몄씠, 利 遺꾪룷 삎깭媛 H3K27ac H3K27me3 궗씠뿉꽌 겕寃 떎由꾩쓣 蹂댁뿬以떎.

Fig. 1. Genome-wide distribution of histone H3K27ac and H3K27me3. (A) Genome browser shows distribution of histone H3K27ac and H3K27me3 in K562 cells. Public K562 ChIP-seq data were obtained from GEO database. Red and pale blue shadows represent H3K27ac- and H3K27me3-enriched regions, respectively. (B) Distribution of H3K27ac and H3K27me3 was visualized using density heatmap at transcription start sites (TSSs) ±5 Kb. TSSs were sorted by H3K27ac or H3K27me3 levels at TSSs ±5 Kb. Density heatmap was expanded to TSSs ±20 Kb with same ordering.

吏湲덇퉴吏 떎뼇븳 醫낅쪟쓽 엳뒪넠 蹂삎씠 諛앺議뚯쑝硫, 蹂삎쓽 醫낅쪟뿉 뵲씪 쑀쟾泥댁뿉꽌 遺꾪룷븯뒗 쐞移섍 떎瑜 寃껋쑝濡 굹궗떎. H3K27ac H3K27me3뒗 겕濡쒕쭏떞 援ъ“뿉 誘몄튂뒗 쁺뼢씠 鍮꾨릺湲 븣臾몄뿉 씠뱾 몢 蹂삎 룞씪븳 겕濡쒕쭏떞 遺쐞뿉꽌 븿猿 씪뼱굹吏 븡쓣 寃껋쑝濡 삁긽릺硫, 떎젣濡 쑀쟾옄 씤빖꽌뿉꽌 씠뱾 엳뒪넠 蹂삎 遺꾪룷쓽 뿭긽愿愿怨꾧 蹂닿퀬릺뿀떎(Wang et al., 2008; Katoh et al., 2018). 삉븳 엳뒪넠 蹂삎쓽 醫낅쪟뿉 뵲씪 遺꾪룷 삎깭룄 떎瑜 寃껋쑝濡 蹂댁씠硫, 씠뒗 Encyclopedia of DNA Elements (ENCODE) 봽濡쒖젥듃쓽 寃곌낵뿉꽌룄 뼵湲됰릺뿀떎(Landt et al., 2012). 뵲씪꽌 H3K27ac H3K27me3쓽 寃쎌슦泥섎읆 洹 遺꾪룷 삎깭媛 떎瑜대㈃ ChIP-seq 뜲씠꽣쓽 깮臾쇱젙蹂댄븰쟻 遺꾩꽍쓣 쐞빐 엳뒪넠 蹂삎씠 씪뼱굹뒗 吏뿭, 利 peak瑜 꽑蹂꾪븷 븣, 遺꾩꽍 諛⑸쾿쓣 떎瑜닿쾶 궗슜빐빞 븷 寃껋씠硫, 씠뒗 쑀쟾泥 닔以뿉꽌 엳뒪넠 蹂삎쓣 젣濡 빐꽍븯湲 쐞빐 以묒슂븳 怨쇱젙쑝濡 깮媛곷맂떎.

H3K27 蹂삎 ChIP-seq 뜲씠꽣뿉꽌 peak 꽑蹂 諛⑸쾿 鍮꾧탳

H3K27ac H3K27me3 ChIP-seq 뜲씠꽣 遺꾩꽍 怨쇱젙뿉꽌 peak瑜 쟻젅븯寃 꽑蹂꾪븯湲 쐞븯뿬 珥 꽭 媛吏 議곌굔쓣 꽑젙븯怨 鍮꾧탳븯떎(Fig. 2A). 媛옣 씪諛섏쟻쑝濡 궗슜릺뒗 遺꾩꽍 룄援ъ씤 MACS2 꼻寃 遺꾪룷븯뒗 엳뒪넠 蹂삎뿉 二쇰줈 궗슜릺뒗 SICER瑜 꽑깮븯쑝硫, MACS2뿉꽌뒗 narrow peaks (narrow) 샃뀡怨 broad peaks (broad) 샃뀡쓣 鍮꾧탳븯떎. 꽑蹂꾨맂 H3K27ac H3K27me3 peaks뒗 紐⑤몢 媛곴컖쓽 엳뒪넠 蹂삎씠 留롮씠 遺꾪룷븯뒗 遺쐞뿉 옄由ы븯怨 엳뿀떎(Fig. 2B). 씠 븣 H3K27ac쓽 寃쎌슦, narrow broad뒗 H3K27ac媛 듅엳 留롮씠 遺꾪룷븯뒗 諛吏 遺쐞瑜 슦꽑쟻쑝濡 peak濡 꽑蹂꾪븯뒗 諛섎㈃ SICER뿉꽌뒗 H3K27ac媛 릺뼱 엳뒗 遺쐞 쟾泥닿 꼻寃 꽑蹂꾨릺뿀떎. 씠뒗 怨좊Ⅴ寃 遺꾪룷븯吏 븡뒗 H3K27ac쓽 遺꾪룷 삎깭뿉 쓽빐 굹굹뒗 李⑥씠濡 異붿젙맂떎. 쑀쟾泥 쟾泥댁뿉꽌 꽑蹂꾨맂 peaks쓽 湲몄씠瑜 鍮꾧탳븯硫, narrow, broad, SICER 諛⑸쾿 닚쑝濡 洹 湲몄씠媛 利앷븯떎(Fig. 2C). Narrow broad濡 꽑蹂꾪븳 H3K27ac peaks쓽 룊洹 湲몄씠뒗 491 bp 1,915 bp쑝硫, 媛숈 諛⑸쾿쑝濡 꽑蹂꾨맂 H3K27me3 peaks쓽 湲몄씠(433 bp 1,686 bp)蹂대떎 議곌툑 湲몄뿀떎. 븯吏留 SICER濡 꽑蹂꾪븷 寃쎌슦, H3K27me3 peaks쓽 룊洹 湲몄씠(6,036 bp)媛 H3K27ac peaks쓽 룊洹 湲몄씠(5,170 bp)蹂대떎 湲몄뿀쑝硫, 씠뒗 SICER뿉 쓽븳 peak 꽑蹂꾩씠 H3K27me3쓽 꼻 遺꾪룷 삎깭瑜 옒 諛섏쁺븯뒗 寃껋쑝濡 빐꽍븷 닔 엳떎. 룞씪븳 ChIP-seq 뜲씠꽣濡쒕꽣 湲몄씠媛 떎瑜닿쾶 꽑蹂꾨맂 peaks쓽 엳뒪넠 蹂삎 닔以쓣 鍮꾧탳븯湲 쐞빐 CPM 媛믪쓣 怨꾩궛븯쑝硫, CPM 媛믪 peaks쓽 湲몄씠 鍮꾨븯뿬 利앷븯떎(Fig. 2D). CPM peak 궡뿉 議댁옱븯뒗 read쓽 닔瑜 쟾泥 read 닔濡 蹂댁젙븳 媛믪쑝濡, peak 옄泥댁쓽 湲몄씠媛 湲몄닔濡 read 닔媛 利앷븯湲 븣臾몄뿉 peak 湲몄씠 CPM 紐⑤몢 쑀궗븳 寃쏀뼢꽦쓣 蹂댁씠뒗 寃껋쑝濡 깮媛곷맂떎.

Fig. 2. Peak calling using MACS2 and SICER tools in public histone H3K27ac and H3K27me3 ChIP-seq data. (A) H3K27ac and H3K27me3 peaks were identified using peak caller MACS2 and SICER with three analysis methods. (B) Peaks identified by each method were presented in genome browser of H3K27ac and H3K27me3 using different colors. Length of peaks (C) and counts per million (CPM) values of peaks (D) were graphed in boxplot. Box and whiskers represent interquartile range (IQR) and ±1.5×IQR values from edge of the box, respectively. (E) Signal-to-background ratio (S/B ratio) was calculated by analyzing H3K27ac or H3K27me3 level at each peak. (F) The S/B ratio of H3K27ac or H3K27me3 peaks was compared between three analysis methods. Difference of the S/B ratio was indicated in multiples.

Peak 꽑蹂 諛⑸쾿쓽 쟻젅꽦쓣 뙆븙븯湲 쐞빐 signal-to-background ratio (S/B ratio)瑜 怨꾩궛븯떎(Fig. 2E). 엳뒪넠 蹂삎씠 留롮씠 遺꾪룷븯뒗 peak瑜 湲곗쑝濡 븯뿬 peak濡 꽑蹂꾨릺吏 븡 遺쐞瑜 non-peak regions쑝濡 援щ텇븳 썑, peak non-peak region 궡뿉 議댁옱븯뒗 read 닔瑜 痢≪젙븯떎. 痢≪젙븳 read 닔濡 peak non-peak region쓽 CPM 媛믪쓣 怨꾩궛븯쑝硫, peaks non-peak regions쓽 룊洹 CPM 媛믪쓣 鍮꾧탳븿쑝濡쒖뜥 S/B ratio瑜 룄異쒗븯떎. 꽭 媛吏 遺꾩꽍 諛⑸쾿쑝濡 꽑蹂꾪븳 peaks뿉꽌 S/B ratio瑜 鍮꾧탳븳 寃곌낵, H3K27ac쓽 narrow broad, 洹몃━怨 narrow SICER 궗씠뿉꽌 洹 鍮꾩쑉 媛곴컖 2諛곗 2.5諛 利앷븯吏留, H3K27me3뒗 媛곴컖 7諛곗 18.8諛 利앷븯떎(Fig. 2F). 씠泥섎읆 H3K27me3 ChIP-seq 뜲씠꽣쓽 S/B ratio媛 遺꾩꽍 諛⑸쾿뿉 뵲씪 겙 李⑥씠瑜 蹂댁씠뒗 寃껋 H3K27ac뿉 鍮꾪빐 H3K27me3 뜲씠꽣媛 peak 꽑蹂 諛⑸쾿뿉 쓽빐 뜑 留롮씠 쁺뼢 諛쏆쓣 닔 엳쓬쓣 떆궗븳떎. 뵲씪꽌 꼻寃 遺꾪룷븯뒗 H3K27me3쓽 peak뒗 MACS2媛 븘땶 SICER瑜 씠슜븯뿬 꽑蹂꾪븯뒗 寃껋씠 쟻젅븳 寃껋쑝濡 깮媛곷맂떎.

H3K27 蹂삎 peak 꽑蹂 諛⑸쾿쓽 쟻젅꽦 寃利

Public K562 ChIP-seq 뜲씠꽣瑜 씠슜븯뿬 鍮꾧탳븳 peak 꽑蹂 諛⑸쾿씠 쟻젅븳吏 솗씤븯湲 쐞빐 蹂 뿰援ъ떎뿉꽌 닔뻾븳 K562 control (Con) H3K27ac ChIP-seq 뜲씠꽣 H3K27me3 ChIP-seq 뜲씠꽣瑜 遺꾩꽍븯떎. 2踰덉쓽 諛섎났 떎뿕쑝濡 뼸 Con ChIP-seq 뜲씠꽣뒗 Spearman 긽愿愿怨 遺꾩꽍뿉꽌 굹궃 寃껋쿂읆 public ChIP-seq 뜲씠꽣 넂 쑀궗꽦쓣 蹂댁떎(Fig. 3A, B). Con ChIP-seq 뜲씠꽣뿉꽌 꽑蹂꾪븳 H3K27ac H3K27me3 peaks뒗 엳뒪넠 蹂삎씠 留롮 遺쐞뿉꽌 굹궗쑝硫, peak쓽 湲몄씠룄 narrow뿉꽌 SICER 닚꽌濡 利앷븯떎(Fig. 3C). Con ChIP-seq 뜲씠꽣쓽 peaks뿉꽌 S/B ratio瑜 鍮꾧탳븳 寃곌낵, H3K27ac뒗 紐⑤뱺 遺꾩꽍 諛⑸쾿뿉꽌 겙 李⑥씠媛 굹吏 븡 諛섎㈃(1.4~2.6諛), H3K27me3뒗 narrow broad, 洹몃━怨 narrow SICER 궗씠뿉꽌 媛곴컖 5.4諛곗 9諛 李⑥씠瑜 蹂댁뿬二쇱뿀떎(Fig. 3D).

Fig. 3. Peak calling in histone H3K27ac and H3K27me3 ChIP-seq data of control K562 cells. H3K27ac and H3K27me3 ChIP-seq were carried out in K562 control (Con) cells with two replicates (R1 and R2). (A) Scatter plots show correlation of public ChIP-seq data with Con R1 and R2 ChIP-seq data with Spearman correlation coefficients. (B) The correlation coefficients were also measured between H3K27ac and H3K27me3 ChIP-seq data and visualized by a heatmap. (C) Genome browser shows peaks identified by three analysis methods in green rectangles. (D) The S/B ratio of H3K27ac and H3K27me3 peaks was compared between three analysis methods with two replicates. Difference of the S/B ratio was indicated in multiples.

꽭 媛吏 諛⑸쾿쑝濡 꽑蹂꾪븳 peaks쓽 S/B ratio뒗 H3K27ac뿉 鍮꾪빐 H3K27me3뿉꽌 겙 李⑥씠瑜 蹂댁쑝硫, 씠윭븳 쁽긽 public 뜲씠꽣 Con 뜲씠꽣 紐⑤몢뿉꽌 굹궗떎. 씠뒗 吏㏃ 湲몄씠뿉 遺꾪룷븯뒗 H3K27ac뿉 鍮꾪빐 湲 嫄곕━뿉 꼻寃 遺꾪룷븯뒗 H3K27me3媛 ChIP-seq 뜲씠꽣 遺꾩꽍 諛⑸쾿뿉 쓽빐 뜑 留롮씠 쁺뼢 諛쏆쓬쓣 쓽誘명븯誘濡 엳뒪넠 蹂삎쓽 遺꾪룷 삎깭뿉 뵲씪 쟻젅븳 peak 꽑蹂 諛⑸쾿쓣 궗슜븯뒗 寃껋씠 븘슂븳 寃 媛숇떎. 븘슱윭 public怨 Con ChIP-seq 뜲씠꽣 紐⑤몢뿉꽌 H3K27ac쓽 S/B ratio媛 H3K27me3蹂대떎 넂븯뒗뜲, 씠뒗 ChIP 떎뿕뿉 궗슜븳 빆泥댁쓽 듅씠꽦怨 愿젴맂 寃껋쑝濡 異붿젙맂떎. Fig. 1A Fig. 3C뿉꽌 蹂댁뿬吏뒗 寃껋쿂읆 H3K27me3 ChIP-seq 뜲씠꽣쓽 noise signal씠 H3K27ac 뜲씠꽣뿉 鍮꾪빐 넂寃 굹굹硫, 넂 noise뒗 寃곌낵쟻쑝濡 non-peak CPM 媛믪쓣 利앷떆궓떎.

Sequencing depth뿉 뵲瑜 H3K27 蹂삎쓽 ChIP-seq 뜲씠꽣 鍮꾧탳

ChIP-seq 씪씠釉뚮윭由щ줈 NGS 떆떛쓣 닔뻾븯硫 씪젙븳 湲몄씠쓽 reads瑜 뼸寃 릺뒗뜲, read쓽 媛쒖닔媛 留롮쓣닔濡 sequencing depth뒗 利앷븳떎. 븯吏留 떆떛 鍮꾩슜怨 遺꾩꽍 떆媛꾩씠 利앷븯뒗 떒젏룄 엳떎. 뵲씪꽌 뜲씠꽣瑜 슚쑉쟻쑝濡 遺꾩꽍븯湲 쐞빐 쟻젅븳 湲몄씠 媛쒖닔쓽 read瑜 뼸뼱빞 븳떎. 遺꾪룷 삎깭媛 떎瑜 H3K27ac H3K27me3쓽 ChIP-seq 뜲씠꽣 遺꾩꽍뿉 쟻젅븳 sequencing depth瑜 븣븘蹂닿린 쐞빐 Con ChIP-seq 뜲씠꽣쓽 쟾泥 read 닔瑜 꽕 媛吏 湲곗(15 M, 30 M, 60 M, 120 M)쑝濡 젣븳븯怨, 씠썑 遺꾩꽍쓣 닔뻾븯떎(Fig. 4A). Genome browser뿉꽌 蹂대㈃ H3K27ac뒗 read 닔 긽愿뾾씠 쑀궗븳 遺꾪룷 삎깭瑜 굹궡吏留, H3K27me3뒗 60 M 씠긽쓽 read 닔뿉꽌 湲멸퀬 洹좎씪븳 遺꾪룷 삎깭瑜 蹂댁뿬以떎(Fig. 4B). Sequencing depth peak 꽑蹂 궗씠쓽 愿怨꾨 議곗궗븯湲 쐞빐 H3K27ac쓽 peak뒗 broad 諛⑸쾿쑝濡, 洹몃━怨 H3K27me3쓽 peak뒗 SICER 諛⑸쾿쑝濡 꽑蹂꾪븯떎. H3K27ac뒗 60 M 120 M reads뿉꽌 30 M뿉 鍮꾪빐 媛곴컖 1.9諛곗 3.6諛 留롮 peaks媛 꽑蹂꾨릺뿀怨, H3K27me3뒗 read 닔 긽愿뾾씠 쑀궗븳 媛쒖닔쓽 peaks媛 꽑蹂꾨릺뿀떎(Fig. 4C). 꽕 媛吏 湲곗쓽 read 닔뿉 븯뿬 peak쓽 S/B ratio瑜 鍮꾧탳븯쓣 븣 H3K27ac H3K27me3뿉꽌 15 M 120 M 궗씠뿉 1.9諛~2.6諛곗쓽 李⑥씠瑜 蹂댁쑝굹, 씠뒗 peak 꽑蹂 諛⑸쾿뿉 븳 S/B ratio쓽 寃⑹감蹂대떎 옉떎(Fig. 4D). H3K27ac뿉꽌 꽑蹂꾨맂 peaks쓽 쐞移섎 鍮꾧탳븯湲 쐞빐 以묒꺽 뿬遺瑜 遺꾩꽍븳 寃곌낵, 60 M 120 M뿉꽌 깉濡쒖슫 peaks媛 留롮씠 異붽릺뿀쑝硫, 듅엳 120 M뿉꽌 700 bp 씠븯쓽 peaks쓽 利앷媛 몢뱶윭吏꾨떎(Fig. 4E). 씠뒗 留롮 read 닔뿉 쓽빐 깉濡 꽑蹂꾨맂 peaks媛 遺遺 吏㏃ 湲몄씠엫쓣 븣 닔 엳떎. 諛섎㈃ H3K27me3뒗 15 M뿉꽌 120 M源뚯 peaks쓽 遺遺꾩씠 以묒꺽릺硫, read 닔媛 利앷븷닔濡 1~4 Kb 궗씠쓽 peaks媛 媛먯냼븯怨, 10~50 Kb 궗씠쓽 peaks媛 利앷븳떎(Fig. 4F). Fig. 4B뿉꽌 read 닔뿉 뵲씪 꽑蹂꾨맂 peaks瑜 H3K27ac H3K27me3쓽 遺꾪룷 닔以怨 鍮꾧탳븯硫, H3K27ac 120 M뿉꽌 깉濡 꽑蹂꾨맂 peaks뒗 蹂삎씠 씪뼱굹뒗 遺쐞瑜 젣濡 諛섏쁺븯吏 紐삵븯吏留 H3K27me3媛 꼻寃 遺꾪룷븯뒗 쁺뿭 120 M뿉꽌 peak濡 쟻젅엳 꽑蹂꾨릺뿀떎. 뵲씪꽌 엳뒪넠 蹂삎쓽 醫낅쪟뿉 뵲씪 넂 sequencing depth媛 삤엳젮 false-positive peak 꽑蹂꾩쓣 珥덈옒븷 닔 엳뒗 寃껋쑝濡 깮媛곷릺硫, H3K27me3泥섎읆 꼻 遺쐞뿉 遺꾪룷븯뒗 엳뒪넠 蹂삎 sequencing depth쓽 利앷媛 쟻젅븳 peak 꽑蹂꾩뿉 湲띿젙쟻쑝濡 옉슜븯뒗 寃껋쑝濡 蹂댁씤떎.

Fig. 4. Analysis of histone H3K27ac and H3K27me3 ChIP-seq data in different sequencing depth. H3K27ac and H3K27me3 ChIP-seq data of control K562 cells (Con) were re-generated with 15, 30, 60 or 120 million (M) read counts. (A) The total read counts of Con ChIP-seq data were listed in a table. (B) Distribution of histone H3K27ac and H3K27me3 and peaks were visualized in genome browser with different read counts. Newly identified peaks in 120 M for H3K27ac were highlighted with red shadow. (C) The number of identified peaks was graphed in 15 M, 30 M, 60 M and 120 M. (D) The S/B ratio of peaks was compared with different read counts. Difference of the S/B ratio was indicated in multiples. Histone H3K27ac (E) and H3K27me3 peaks (F) identified in different read counts were overlapped and visualized by venn diagram (left). The number of peaks was graphed according to the length of them (right).

蹂 뿰援ъ뿉꽌 遺꾩꽍븳 Con ChIP-seq 뜲씠꽣뒗 paired-end 떆떛쓣 넻빐 솗蹂댄븳 뜲씠꽣濡, 2媛쒖쓽 reads媛 1媛쒖쓽 fragment뿉꽌 뼸뼱吏湲 븣臾몄뿉 Fig. 4뿉꽌 뼵湲됲븳 15~ 120 M쓽 read 닔뒗 7.5~60 M쓽 fragments瑜 諛섏쁺븳떎. 洹몃윭굹 single-end 떆떛쑝濡 뼸 ChIP-seq 뜲씠꽣뒗 1媛쒖쓽 read媛 1媛쒖쓽 fragment瑜 諛섏쁺븯誘濡 NGS 뜲씠꽣쓽 sequencing depth뒗 떆떛 諛⑸쾿뿉 뵲씪 鍮꾧탳빐빞 븳떎. ENCODE 媛씠뱶씪씤뿉 뵲瑜대㈃ mammalian cells뿉꽌 쑀쓽誘명븳 ChIP-seq 뜲씠꽣瑜 솗蹂댄븯湲 쐞빐 理쒖냼 1~2泥쒕쭔媛(10~20 M) 씠긽쓽 uniquely mapped reads媛 븘슂븯떎(Landt et al., 2012). 꼻寃 遺꾪룷븯뒗 엳뒪넠 蹂삎쓽 寃쎌슦 sequencing depth媛 넂쓣닔濡 뜲씠꽣쓽 吏덉쟻 닔以씠 利앷븯뒗 寃쏀뼢쓣 蹂댁뒗뜲, 씠뒗 sequencing depth뿉 뵲씪 떎뼇븳 엳뒪넠 蹂삎쓽 ChIP-seq 뜲씠꽣瑜 鍮꾧탳븳 뿰援 寃곌낵 씪移섑븳떎(Jeon et al., 2020). 뵲씪꽌 醫곴쾶 遺꾪룷븯뒗 H3K27ac뒗 paired-end 湲곗 30 M쓽 reads留 뼸뼱룄 쑀쓽誘명븳 ChIP-seq 뜲씠꽣瑜 솗蹂댄븷 닔 엳吏留, 꼻寃 遺꾪룷븯뒗 H3K27me3뒗 쟻뼱룄 60 M 씠긽쓽 reads媛 븘슂븳 寃껋쑝濡 깮媛곷맂떎. 삉븳 read 닔뿉 뵲瑜 S/B ratio 李⑥씠뒗 peak 꽑蹂 諛⑸쾿뿉 뵲瑜 李⑥씠蹂대떎 옉寃 굹궗쑝硫, H3K27ac 120 M뿉꽌 S/B ratio媛 15 M蹂대떎 2.6諛 넂븯吏留 遺쟻젅븳 peaks媛 븿猿 꽑蹂꾨릺뿀뜕 寃껋쑝濡 蹂댁븘 S/B ratio뒗 뜲씠꽣 옄泥댁쓽 吏덉쟻 닔以 鍮꾧탳蹂대떎뒗 遺꾩꽍 諛⑸쾿쓽 鍮꾧탳뿉 솢슜븯뒗 寃껋씠 쟻젅븯떎怨 궗猷뚮맂떎.

蹂 뿰援 寃곌낵뒗 쑀쟾泥 닔以뿉꽌 엳뒪넠 H3K27ac H3K27me3쓽 遺꾪룷 湲몄씠媛 떎瑜대ʼn, 꼻寃 遺꾪룷븯뒗 H3K27me3뒗 醫곴쾶 씪뼱굹뒗 H3K27ac뿉 鍮꾪빐 peaks 꽑蹂 諛⑸쾿씠 以묒슂븿쓣 蹂댁뿬以떎. 삉븳 sequencing depth媛 H3K27ac쓽 뜲씠꽣 遺꾩꽍뿉 蹂 쁺뼢쓣 誘몄튂吏 븡吏留, 꼫臾 넂 sequencing depth뒗 peak 꽑蹂 怨쇱젙뿉꽌 遺젙쟻씤 쁺뼢쓣 誘몄튂뒗 寃껋쑝濡 굹궗떎. 諛섎㈃ H3K27me3뒗 sequencing depth媛 넂쓣닔濡 ChIP-seq 뜲씠꽣쓽 吏덉쟻 닔以씠 利앷븯떎. 뵲씪꽌 엳뒪넠 蹂삎뿉 븳 ChIP-seq 떎뿕쓣 닔뻾븷 븣 엳뒪넠 蹂삎씠 遺꾪룷븯뒗 삎깭瑜 怨좊젮븯뿬 sequencing depth瑜 寃곗젙븯怨, 遺꾩꽍 諛⑸쾿쓣 꽑깮븯뒗 寃껋씠 븘슂븯떎怨 뿬寃⑥쭊떎. 씠젃寃 뼸뼱吏 寃곌낵뒗 젙솗븳 깮臾쇳븰쟻 젙蹂대 젣怨듯븯怨 굹븘媛 吏덈퀝쓽 吏꾨떒 諛 移섎즺젣 媛쒕컻뿉 솢슜맆 닔 엳쓣 寃껋씠떎.

Abbreviations
15~120 M

15 million-120 million

ChIP

Chromatin immuniprecipitation

ChIP-seq

Chromatin굀mmuniprecipitation-sequencing

Con

Control

CPM

Counts per million

ENCODE

Encyclopedia of DNA elements

EZH2

Enhancer of zeste 2 polycomb epressive complex 2

GEO

Gene expression omnibus

H3K27ac

Histone H3K27 acetylation

H3K27me3

Histone H3K27 tri-methylation

H3K36me3

Histone H3K36 tri-methylation

H3K4me1

Histone H3K4 mono-methylation

H3K4me3

Histone H3K4 tri-methylation

H3K9me3

Histone H3K9 tri-methylation

HDAC

Histone deacetylase

IGV

Integrative genomics viewer

MAPQ

Mapping quality

MNase

Micrococcal nuclease

NGS

Next-generation sequencing

S/B ratio

Signal-to-background ratio

TE

Tris-EDTA

TSSs

Transcription start sites

ACKNOWLEDGEMENT

This study was supported by the 랡K21 Four Program of Pusan National University.

CONFLICT OF INTEREST

The authors have declared no conflict of interest.

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