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Kev Sib Cuam Tshuam ntawm Chromatin raws li Hi-C
Hi-C yog ib txoj kev tsim los ntes cov qauv genomic los ntawm kev sib txuas ua ke probing proximity-based interactions thiab high-throughput sequencing. Txoj kev no yog raws li chromatin crosslinking nrog formaldehyde, ua raws li kev zom thiab rov ua dua tshiab hauv txoj kev uas tsuas yog cov khoom uas tau txuas nrog covalently thiaj li yuav tsim cov khoom ligation. Los ntawm kev sequencing cov khoom ligation no, nws muaj peev xwm kawm txog 3D lub koom haum ntawm genome. Hi-C ua rau kev kawm txog kev faib tawm ntawm cov feem ntawm genome uas tau ntim me me (A compartments, euchromatin) thiab yuav muaj kev ua haujlwm transcriptional, thiab cov cheeb tsam uas tau ntim nruj dua (B compartments, Heterochromatin). Hi-C kuj tseem siv tau los taw qhia Topologically Associated Domains (TADs), cov cheeb tsam ntawm genome uas muaj cov qauv tais thiab yuav muaj cov qauv qhia zoo sib xws, thiab los txheeb xyuas cov chromatin loops, DNA cheeb tsam uas tau anchored ua ke los ntawm cov protein thiab uas feem ntau muaj ntau yam hauv cov ntsiab lus tswj hwm. BMKGene qhov kev pabcuam Hi-C sequencing ua rau cov kws tshawb fawb muaj peev xwm tshawb nrhiav qhov chaw ntawm genomics, qhib txoj hauv kev tshiab rau kev nkag siab txog kev tswj hwm genome thiab nws cov txiaj ntsig rau kev noj qab haus huv thiab kab mob.
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Kev Tshawb Fawb Txog Chromatin Immunoprecipitation (ChIP-seq)
Chromatin Immunoprecipitation (CHIP) yog ib txoj kev siv cov tshuaj tua kab mob los xaiv cov protein uas khi DNA thiab lawv cov hom phiaj genomics sib xws. Nws txoj kev koom ua ke nrog NGS ua rau muaj kev txheeb xyuas thoob plaws genome ntawm cov hom phiaj DNA uas cuam tshuam nrog kev hloov kho histone, cov yam ntxwv sau ntawv, thiab lwm cov protein uas khi DNA. Txoj hauv kev dynamic no ua rau muaj kev sib piv ntawm cov chaw khi thoob plaws ntau hom cell, cov ntaub so ntswg, lossis cov mob. Cov ntawv thov ChIP-Seq suav nrog kev kawm txog kev tswj hwm kev sau ntawv thiab txoj hauv kev loj hlob mus rau kev piav qhia txog cov txheej txheem kab mob, ua rau nws yog ib qho cuab yeej tseem ceeb rau kev nkag siab txog cov toj roob hauv pes tswj hwm genomic thiab kev txhim kho kev nkag siab kho mob.
Platform: Illumina NovaSeq
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Kev txheeb xyuas tag nrho cov noob caj noob ces bisulfite (WGBS)
Cov txheej txheem no, raws li kev kho mob bisulfite ntawm DNA los ua kom muaj kev hloov pauv ntawm cov cytosines unmethylated mus rau uracil (C rau U), thaum tawm hauv cov cytosines methylated tsis hloov pauv, sawv cev rau txoj kev ntsuas kub rau kev tshawb nrhiav tob ntawm DNA methylation, tshwj xeeb tshaj yog qhov chaw thib tsib hauv cytosine (5-mC), tus tswj hwm tseem ceeb ntawm kev qhia tawm gene thiab kev ua haujlwm ntawm tes.
Cov txheej txheem no muab kev daws teeb meem ib leeg, uas tso cai rau cov kws tshawb fawb tshawb nrhiav cov methylome thiab nrhiav pom cov qauv methylation tsis zoo hauv cov qauv kuaj. Los ntawm kev siv WGBS, cov kws tshawb fawb tuaj yeem tau txais kev nkag siab tsis zoo txog cov toj roob hauv pes methylation thoob plaws genome, muab kev nkag siab zoo txog cov txheej txheem epigenetic uas yog lub hauv paus ntawm ntau yam txheej txheem biological thiab kab mob.
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Kev Ntsuas rau Transposase-Accessible Chromatin nrog High Throughput Sequencing (ATAC-seq)
ATAC-seq yog ib txoj kev siv high-throughput sequencing siv rau kev tshuaj xyuas chromatin thoob plaws genome. Nws siv muab kev nkag siab tob dua txog cov txheej txheem nyuaj ntawm kev tswj hwm epigenetic thoob ntiaj teb ntawm kev qhia tawm gene. Txoj kev siv hyperactive Tn5 transposase los ua ke fragment thiab tag cov cheeb tsam chromatin qhib los ntawm kev ntxig cov sequencing adapters. Tom qab ntawd PCR amplification ua rau muaj kev tsim cov tsev qiv ntawv sequencing, uas tso cai rau kev txheeb xyuas cov cheeb tsam chromatin qhib nyob rau hauv cov xwm txheej tshwj xeeb. ATAC-seq muab kev pom holistic ntawm cov toj roob hauv pes chromatin nkag tau, tsis zoo li cov txheej txheem uas tsuas yog tsom mus rau cov chaw khi transcription factor lossis cov cheeb tsam hloov kho histone tshwj xeeb. Los ntawm kev sequencing cov cheeb tsam chromatin qhib no, ATAC-seq qhia cov cheeb tsam uas yuav muaj cov kab ke tswj hwm thiab cov chaw khi transcription factor, muab kev nkag siab zoo rau kev hloov pauv dynamic ntawm kev qhia tawm gene thoob plaws genome.
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Kev Txheeb Xyuas Bisulfite Sequencing (RRBS) Uas Txo Qhov Sawv Cev
Kev txo qis kev sawv cev bisulfite sequencing (RRBS) vam khom kev nplua nuj ntawm CpG kob-nplua nuj thaj chaw los ntawm MspI cleavage ua raws li kev xaiv qhov loj ntawm 200-500/600 bps fragments. Yog li ntawd, tsuas yog thaj chaw ze rau CpG kob raug sequenced, thaum cov uas muaj CpG kob deb raug tshem tawm ntawm kev tshuaj xyuas. Cov txheej txheem no, ua ke nrog bisulfite sequencing, tso cai rau kev kuaj pom siab ntawm DNA methylation, thiab txoj hauv kev sequencing, PE150, tsom mus rau qhov kawg ntawm cov ntxig es tsis yog nruab nrab, ua rau muaj kev ua haujlwm ntawm methylation profiling.
Cov txheej txheem no tau tshwm sim los ua lwm txoj hauv kev pheej yig thiab ua tau zoo rau Whole Genome Bisulfite Sequencing (WGBS) hauv kev tshawb fawb DNA methylation. Thaum WGBS muab kev nkag siab dav dav los ntawm kev tshuaj xyuas tag nrho cov genome ntawm qhov kev daws teeb meem ib qho, nws tus nqi siab tuaj yeem yog qhov txwv, uas RRBS ua kom txo qhov teeb meem no los ntawm kev xaiv kev tshuaj xyuas ib feem ntawm cov genome. Txoj kev no yog ib qho cuab yeej muaj nqis uas ua rau muaj kev tshawb fawb DNA methylation pheej yig thiab txhim kho kev paub txog cov txheej txheem epigenetic.
