Gli autori sono supportati da A * STAR di Singapore. Inoltre, MJF è supportata da una borsa di studio STAR A * National Science, uno Oreal For Women in Science Fellowship nazionale e Lee Kuan Yew Post-Doctoral Fellowship. YR è supportato da sovvenzioni NIH Codifica HG004456 (R01-01 e R01 HG003521-01). Gli autori riconoscono anche la squadra videografia di 8 Pixel Productions, Singapore, in particolare il signor Kelvin Issey, Mr. Chang Kai Xiang e il signor Sherwin Gan per girare le scene, la signora Siti Rahim per il video editing e la signora Michelle Teo per la voice-over.

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Chia-PET è un metodo sviluppato per identificare le interazioni a lungo raggio di regolazione della trascrizione. Uno dei fattori critici che determinano la qualità di un Chia-PET libreria è la qualità del materiale ChIP. Il protocollo mostrato nel video incorpora l&#39;uso di EGS e formaldeide per reticolare le cellule. L&#39;uso di formaldeide combinato con un secondo reagente di reticolazione che porta un braccio più lungo distanziatore può aiutare nel legame di proteine ​​che non poteva essere vincolati dalla sola formaldeide 3,11,15. Abbiamo costruito librerie con questo metodo, che hanno dimostrato robuste siti di legame e interazioni a lungo raggio 9. Tuttavia, la reticolazione e condizioni chip dovrebbe essere ottimizzato per ogni fattore di interesse, ed è importante non troppo reticolazione come troppo reticolazione comporta difficoltà di frammentazione per sonicazione, e può eventualmente provocare interazioni cromatina spuri . Le interazioni della cromatinaidentificato da Chia-PET dovrebbe essere convalidato con un metodo diverso, come fluorescenza ibridazione in situ 4. Si consiglia un minimo di 100 ng di materiale cromatina. Mentre abbiamo costruito librerie di buona qualità da 50 ng di materiale cromatina, abbiamo osservato che grandi quantità di materiale di partenza ha permesso la costruzione di Chia-PET librerie con meno di 16 cicli di PCR, amplificati in modo da minimizzare e la ridondanza di ogni libreria. Questa ridondanza inferiore correlato con alti tag unici mappati e anche una percentuale elevata di dati utilizzabili, consentendo così una più completa mappa interazione cromatina con meno corsie di sequenziamento. Il volume finale ricco di perle in ogni tubo dovrebbe essere di 50 ul e 100 microlitri per biglie magnetiche e Sepharose rispettivamente. Se il volume ricco di cordone è inferiore a quella indicata, portare a volume minimo imballato con biglie magnetiche o Sepharose simile pre-autorizzato in bianco per ridurre al minimo la perdita di DNA-cuscinetto tallones nei passaggi successivi. Sawed-off consigli o suggerimenti di grandi dimensioni-core dovrebbe essere usato per pipettaggio perline Sepharose. Le modifiche seguenti sono stati inseriti a seguito della precedentemente pubblicato con il Chia-PET protocollo 5. In primo luogo, magnetici perle G sono stati utilizzati per ridurre al minimo la perdita del campione durante i lavaggi. Inoltre, abbiamo identificato non-specifici gruppi con dimensioni approssimative di 100 bp e 138 bp per essere ampliconi di auto-legato mezze linkers o / e adattatori. Pertanto, si riduce la concentrazione di biotinilati mezze linkers e 454 GS20 adattatori per ridurre al minimo non-specifiche bande durante l&#39;amplificazione PCR. Il volume vicinanza ligazione è stato ridotto da 50 ml a 10 ml per minimizzare la perdita di campione durante successive fasi di purificazione e di risparmiare sui costi dei reagenti. Abbiamo anche aumentato il tempo di incubazione di immobilizzare Chia-PET DNA a microsfere per garantire la cattura massima di Chia-PET DNA sulle perline streptavidina. Durante la fase di legatura di prossimità, legature chimerici that non rappresentano vero in vivo interazioni cromatina sono inevitabilmente generate in modo non specifico e casuale. Quindi, per valutare la qualità dei dati da qualsiasi Chia-PET esperimento, il tasso di chimerismo viene stimata dalla uso di due differenti mezze linkers nucleotidica specifica con codici a barre e TAAG ATGT 5. Dopo alta sequenziamento, il Chia-PET sequenze sono dapprima analizzati per la composizione barcode linker e sequenze derivate da prodotti di ligazione specifici e non specifici prodotti di ligazione si possono distinguere 8. La percentuale di chimere conosciute (ad esempio eterodimeri linkers AB) presenti nella nostra società in-house MCF-7 RNA polimerasi II Chia-PET librerie è inferiore al 15%. Chia-PET sequenze sono poi classificati in due categorie, vale a dire auto-legatura tra PET e PET-legatura. Self-legatura PET sono ottenuti da self-circolarizzazione legatura dei frammenti di cromatina, mentre inter-legatura PET sono derivati ​​da inter-ligazione tra due frammenti di DNA differenti. Quest&#39;ultimo è quindi suddiviso in tre differenti categorie a seconda della distanza genomica di ciascuna etichetta sul cromosoma stesso (intracromosomica inter-ligazione PET) o che entrambi i tag vengono mappati a due cromosomi diversi (interchromosomal inter-ligazione PET). Abbiamo sviluppato una Chia-PET pacchetto software strumento per risolvere le diverse categorie 8. Questo si basa sui frammenti di DNA che sono nella libreria. In generale, i frammenti più piccoli ChIP darà una maggiore risoluzione e la tagliata di questi RNA polimerasi II Chia-PET librerie è di circa 4 kb. Inoltre, le interazioni cromatina reali possono essere distinte dal rumore casuale contando il numero di inter-ligazione PET in un cluster interazione, in altre parole, un cluster di conteggio PET alto è detto avere una probabilità maggiore di essere una interazione cromatina reale 8 . Per filtrare i falsi positivi unopassare da ancore altamente arricchito, che può formare inter-legatura PET per caso a caso, un quadro di analisi statistica è stata formulata per spiegare la formazione casuale di qualsiasi inter-legatura animali da compagnia tra due ancore 8. In conclusione, il Chia-PET tecnica permette di mappare le reti di interazione cromatina su scala globale. L&#39;implementazione di ChIP in Chia-PET consente la riduzione della complessità della libreria e rumore di fondo. Inoltre, aggiunge ChIP specificità per le interazioni della cromatina, consentendo l&#39;esame delle interazioni della cromatina specifici associati a particolari fattori di trascrizione 5.

<table border="1"><tbody><tr><td> Nome del reattivo </td><td> Azienda </td><td> Numero di catalogo </td><td> Comments </td></tr><tr><td> 4-20% di pendenza TBE gel </td><td> Invitrogen </td><td> EC6225BOX </td><td> Fase B, 6.3 </td></tr><tr><td> 5 x T4 DNA ligasi Buffer con PEG </td><td> Invitrogen </td><td> 46300018 </td><td> Fase B, 2.2 </td></tr><tr><td> 6% TBE gel </td><td> Invitrogen </td><td> EC6263BOX </td><td> Fase B, 6.8 </td></tr><tr><td> Agilent DNA 100 Assay </td><td> Agilent Technologies </td><td> 5067-1504 </td><td rowspan="3"> Fase B, 7.1 </td></tr><tr><td> Agilent ad alta sensibilità del test del DNA </td><td> Agilent Technologies </td><td> 5067-4626 </td></tr><tr><td> Agilent Bioanalyzer 2100 </td><td> Agilent Technologies </td><td> G2940CA </td></tr><tr><td> Centrifugare Filtri tubi (Spin-X) </td><td> Corning &lt;/ Td&gt; <td> CLS8160 </td><td> Fase B, 3.7 e 6.9 </td></tr><tr><td> Reader Transilluminatore scuro </td><td> Clare Chemical Research </td><td> DR46B </td><td> Fase B, 6.8 </td></tr><tr><td> Digital Sonifier cella Disrupter </td><td> Branson </td><td> 450D-0101063591 </td><td> Fase A, 4,3 </td></tr><tr><td> DynaMag-2 magnete (Concentrator particelle magnetiche) </td><td> Invitrogen </td><td> 123-21D </td><td> Fase A, Fase B 7.5.1, per tutte le biglie magnetiche lavaggio passi </td></tr><tr><td> DynaMag-15 Magnet (Concentrator particelle magnetiche) </td><td> Invitrogen </td><td> 123.01D </td><td> Fase A, 5 e 6 </td></tr><tr><td> DynaMag-PCR (Concentrator particelle magnetiche) </td><td> Invitrogen </td><td> 49-2025 </td><td> Fase B, 6.5 </td></tr><tr><td> Escherichia coli DNA polimerasi I </td><td> NEB </td><td> M0209 </td><td> Fase B, 5,6 </td></tr><tr><td> GlycoBlue </td><td> Ambion </td><td> AM9516 </td&gt; <td> Fase A, Fase B 7.5.1, 4.3 e 6.5 </td></tr><tr><td> Illumina CBOT System Cluster Generation </td><td> Illumina </td><td> SY-301-2002 </td><td> Fase B, 7.4 </td></tr><tr><td> Illumina Genome Analyzer IIx </td><td> Illumina </td><td> SY-301-1301 </td><td> Fase B, 7,5 </td></tr><tr><td> Illumina PE primer </td><td> Illumina </td><td> PE-102-1004 </td><td> Fase B, 5,4 (se necessario) </td></tr><tr><td> Intelli-Mixer </td><td> Palico Biotech </td><td> RM-2L </td><td> Fase B, le incubazioni con rotazione </td></tr><tr><td> LightCycler 480 Real-Time PCR System </td><td> Roche </td><td> 04 640 268 001 </td><td> Fase A, Fase B 7.5.3, 7.3 </td></tr><tr><td> LightCycler480 DNA SYBR Green I MasterMix </td><td> Roche </td><td> 03 752 186 001 </td><td> Fase B, 7.5.3 </td></tr><tr><td> M-280 streptavidina Dynabeads </td><td> Invitrogen </td><td> 11206D </td><td> Fase B, 5.2 </td></tr> &lt;tr&gt; <td> Magnetic (Dynabeads) Proteine ​​G </td><td> Invitrogen </td><td> 100.03D </td><td> Fase A, 5.1.1 e 5.2.1 </td></tr><tr><td> MaXtract ad alta densità (2 ml) </td><td> Qiagen </td><td> 129056 </td><td> Fase A, 7.5.1 </td></tr><tr><td> MaXtract ad alta densità (50ml) </td><td> Qiagen </td><td> 129073 </td><td> Fase B, 4.2 </td></tr><tr><td> MmeI </td><td> NEB </td><td> R0637 </td><td> R0637 </td></tr><tr><td> RNAsi ONE ribonucleasi </td><td> Promega </td><td> M426C </td><td> Fase B, 4.8 </td></tr><tr><td> RNA polimerasi II (8WG16) anticorpo monoclonale </td><td> Covance </td><td> MMS-126R </td><td> Fase A, 5.2.4 </td></tr><tr><td> Provette da centrifuga Oak Ridge (polipropilene) </td><td> Nalgene </td><td> 3119-0050 </td><td> Fase A, 3,1 </td></tr><tr><td> Provette da centrifuga Oak Ridge (Teflon FEP) </td><td> Nalgene </td><td> 3114-0050 </td><td> Fase B, 4.3 </td></tr><tr><td> PHUsion ad alta fedeltà Master Mix </td><td> Finnzymes </td><td> F-531 </td><td> Fase B, 6 </td></tr><tr><td> Picogreen (Quant-iT) reagente dsDNA </td><td> Invitrogen </td><td> P11495 </td><td> Fase A, 7.5.2 </td></tr><tr><td> Polistirolo Provetta a fondo sferico </td><td> BD Biosciences </td><td> 352057 </td><td> Fase A, 4,1 </td></tr><tr><td> Cocktail di inibitori della proteasi compresse (completo, EDTA-free) </td><td> Roche </td><td> 11873580001 </td><td> Fase A, 1.6 in poi </td></tr><tr><td> Proteinasi K Solution (20mg/ml) </td><td> Fermentas </td><td> E00491 </td><td> Fase A, 4.4, 7.5.1 Fase B, 4.1 </td></tr><tr><td> DNA ligasi T4 </td><td> Fermentas </td><td> EL0013 </td><td> Fase B, 2.2, 3.9 e 5.4 </td></tr><tr><td> T4 DNA ligasi Buffer (NEB) </td><td> NEB </td><td> B0202S </td><td> Fase B, 3.3 e 3.9 </td></tr><tr><td> DNA polimerasi T4 </td><td> Promega </td><td> M4215 </td><td>Fase B, 1.2 </td></tr><tr><td> DNA T4 polinucleotide chinasi </td><td> NEB </td><td> M0201 </td><td> Fase B, 3.3 </td></tr><tr><td> TruSeq SBS Kit v5-GA </td><td> Illumina </td><td> FC-104-5001 </td><td> Reggenti per Illumina Genome Analyzer IIx sistema </td></tr><tr><td> TruSeq PE Cluster Kit v2-CBOT-GA </td><td> Illumina </td><td> PE-300-2001 </td><td> per essere utilizzato con il sistema Illumina Generation Cluster CBOT </td></tr><tr><td> SYBR Green I </td><td> Invitrogen </td><td> S-7585 </td><td> Fase B, 6.3 e 6.8 </td></tr><tr><td> XCell SureLock Mini-Cell Electrophoresis sistema </td><td> Invitrogen </td><td> EI0001 </td><td> Fase B, 6.3 e 6.8 </td></tr></tbody></table><table border="1"><tbody><tr><td> Nome </td><td colspan="2"> Sequenza </td><td> Comments </td></tr><tr><td rowspan="2"> Biotinilato mezze linkers A (200ng/μl) </td><td> Top </td><td> 5 &#39;GG CCG CGA / iBiodT / ATC TTA TCC AAC 3&#39; </td><td> 250 nmoli scala HPLC purificato interno Biotin dT (9) </td></tr><tr><td> Bot </td><td> 5 &#39;GTT GGA TAA GAT ATC GC 3&#39; </td><td> 250 nmoli scala HPLC purificato </td></tr><tr><td rowspan="2"> Biotinilato mezze linkers B (200ng/μl) </td><td> Top </td><td> 5 &#39;GGC CGC GA / iBiodT / ATA CAT TCC AAC 3&#39; </td><td> 250 nmoli scala HPLC purificato interno Biotin dT (9) </td></tr><tr><td> Bot </td><td> 5 &#39;GTT GGA ATG TAT ATC GC 3&#39; </td><td> 250 nmoli scala HPLC purificato </td></tr><tr><td rowspan="2"> Non biotinilati mezze linkers (200ng/μl) </td><td> Top </td><td> 5 &#39;GGC CGC GAT ATC GGA TCC AAC 3&#39; </td><td> 250 nmoli scala PCR Grade </td></tr><tr><td> Bot </td><td> 5 &#39;GTT GGA TCC GAT ATC GC 3&#39; </td><td> 250 nmoli scala PCR Grade </td></tr><tr><td rowspan="2"> GS20 Adaptor A (200ng/μl) </td><td> Top </td><td> 5 &#39;CAT TCT CCA CCC TGC GTG TCC CATCTG TTC CCT CCC TGT CTC AGN N 3 &#39; </td><td> 250 nmoli scala PCR Grade </td></tr><tr><td> Bot </td><td> 5&#39;CTG AGA CAG GGA GGG AAC AGA TGG GAC ACG CAG GGA TGA GAT GG 3 &#39; </td><td> 250 nmoli scala PCR Grade </td></tr><tr><td rowspan="2"> GS20 Adattatore B (200ng/μl) </td><td> Top </td><td> 5 &#39;CTG AGA CAC GCA ACA GGG GAT AGG CAA GGC ACA CAG GGG ATA GG 3&#39; </td><td> 250 nmoli scala PCR Grade </td></tr><tr><td> Bot </td><td> 5 &#39;ATC CCC CCT TGT GTG CCT TGC CTA GTT TCC CCT GCG TGT CTC AGN N 3&#39; </td><td> 250 nmoli scala PCR Grade </td></tr><tr><td rowspan="2"> Illumina-NN adattatori </td><td> Top </td><td> 5 &#39;ACA CTC TTT CCC TAC ACG ACG CTC TTC CGA TC 3&#39; </td><td> 250 nmoli scala HPLC purificato Vedi Fase B, 5.4 </td></tr><tr><td> Bot </td><td> 5 &#39;phos - GAT CGG AAG AGC GGT TCA GCA GGA ATG CCG AG 3&#39; </td><td> 250 nmoli scala HPLC purificato fosforilata 5 &#39;Vedere B Step, 5,4 </td></tr><tr><td> Illumina 1-454 (in avanti) primer (10 pM) </td><td colspan="2"> 5 &#39;AAT GAT ACG GCG ACC ACC ACC GAG ATC TAC CTA TCC CCT GTG TGC CTT G 3&#39; </td><td> 250 nmoli scala PCR Grade </td></tr><tr><td> Illumina 2-454 (reverse) primer (10 micron) </td><td colspan="2"> 5 &#39;CAA GCA GAA GAC GGC ATA CGA GAT CGG TCC ATC TCA TCC CTG CGT GTC 3&#39; </td><td> 250 nmoli scala PCR Grade </td></tr><tr><td> qPCR Primer 1.1 (10 pM) </td><td colspan="2"> 5 &#39;AAT GAT ACG GCG ACC ACC GAG A 3&#39; </td><td> 10nmole scala PCR Grade </td></tr><tr><td> qPCR Primer 2.1 (10 pM) </td><td colspan="2"> 5 &#39;CAA GCA GAA GAC GGC ATA CGA 3&#39; </td><td> 10nmole scala PCR Grade </td></tr><tr><td> Illumina 3-454 sequenziamento primer (100 um) </td><td colspan="2"> 5&#39;TGC GTG TCC CAT CTG TTC CCT CCC TGT CTC AG 3 &#39; </td><td> 100nmole scala HPLC purificato </td></tr><tr><td> Illumina 4-454 sequenziamento primer (100 um) </td><td colspan="2"> 5&#39;GTG CCT TGC CTA GTT TCC CCT GCG TGT CTC AG 3 &#39; </td><td> Scala 100nmoleHPLC purificato </td></tr></tbody></table> Oligo ordine Tabella di oligo e adattatori. Da Integrated DNA Technologies (IDT) e preparare linkers e adattatori nello stesso modo descritto in precedenza 10. Mezze linker e adattatori possono essere preparate in anticipo e conservati per diversi mesi a -20 ° C.

chromatin interaction analysis with paired-end tag sequencing (chia-pet) for mapping chromatin interactions and understanding transcription regulation

Genomi sono organizzati in strutture tridimensionali, adottando ordine superiore conformazioni all&#39;interno degli spazi nucleari dimensioni micron 7, 2, 12. Architetture di questo tipo non sono casuali e comportano interazioni tra geni promotori e gli elementi normativi 13. Il legame di fattori di trascrizione specifici per sequenze regolatrici determina una rete di regolazione della trascrizione e coordinamento 1, 14. Interaction Analysis cromatina per tag Sequencing paired-End (Chia-PET) è stato sviluppato per identificare queste strutture sovra-ordinate cromatina 5,6. Cellule vengono fissate e loci interagenti sono catturati dal covalenti DNA-proteina legami incrociati. Per minimizzare il rumore non specifico e ridurre la complessità, così come per aumentare la specificità del analisi dell&#39;interazione cromatina, immunoprecipitazione della cromatina (ChIP) viene usato contro fattori proteici specifici per arricchire frammenti cromatina di interesse prima legatura prossimità. Legatura coinvolgendo mezze linkers successivamente forma legami covalenti tra coppie di frammenti di DNA legati insieme all&#39;interno di complessi cromatina individuali. I accompagnamento MmeI siti di restrizione degli enzimi nella mezza linkers permettono l&#39;estrazione di coppie di terminali tag-linker-tag costrutti (PET) su MmeI digestione. Poiché le mezze linkers sono biotinilati, questi costrutti PET sono purificati utilizzando streptavidina-perle magnetiche. Le PET purificati vengono legati con la prossima generazione di adattatori di sequenziamento e un catalogo di frammenti interagenti viene generato tramite sequencer di nuova generazione come il Illumina Genome Analyzer. Mappatura e analisi bioinformatica viene quindi eseguita per identificare ChIP arricchito vincolante siti e ChIP arricchiti interazioni cromatina 8. Abbiamo prodotto un video per dimostrare aspetti critici del Chia-PET protocollo, in particolare la preparazione del chip come la qualità di ChIP gioca un ruolo importante nel risultato di una Chia-PET libreria. Poiché i protocolli sonomolto lungo, solo i punti critici sono mostrati nel video.

Watch this Scientific Journal Video about Chromatin Interaction Analysis with Paired-End Tag Sequencing ChIA-PET for Mapping Chromatin Interactions and Understanding Transcription Regulation at JoVE.c

Chromatin Interaction Analysis with Paired-End Tag Sequencing ChIA-PET for Mapping Chromatin Interactions and Understanding Transcription Regulation

Interaction Analysis cromatina per tag Sequencing paired-End (Chia-PET) è un metodo per<em&gt; De novo</emRilevazione&gt; delle interazioni della cromatina, per una migliore comprensione del controllo trascrizionale.

Cromatina Analisi Interazioni con Tag Sequencing appaiati-End (Chia-PET) per la mappatura Interazioni cromatina e comprensione del regolamento Trascrizione

Genomes are organized into three-dimensional structures, adopting higher-order conformations inside the micron-sized nuclear spaces 7, 2, 12. Such ...

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Research

JoVE Journal

Biology

Chromatin Interaction Analysis with Paired-End Tag Sequencing (ChIA-PET) for Mapping Chromatin Interactions and Understanding Transcription Regulation

30.5K Views.  Agency for Science, Technology and Research, Singapore. Interaction Analysis cromatina per tag Sequencing paired-End (Chia-PET) è un metodo per De novo delle interazioni della cromatina, per una migliore comprensione del controllo trascrizionale.

Video: Chromatin Interaction Analysis with Paired-End Tag Sequencing ChIA-PET for Mapping Chromatin Interactions and Understanding Transcription Regulation

A Chromatin Assay for Human Brain Tissue

Chronic neuropsychiatric illnesses such as schizophrenia, bipolar disease and autism are thought to result from a combination of genetic and environmental factors that might result in epigenetic alterations of gene expression and other molecular pathology. Traditionally, however, expression studies in postmortem brain were confined to quantification of mRNA or protein. The limitations encountered in postmortem brain research   such as variabilities in autolysis time and tissue integrities   are also likely to impact any studies of higher order chromatin structures. However, the nucleosomal organization of genomic DNA   including DNA:core histone binding - appears to be largely preserved in representative samples provided by various brain banks. Therefore, it is possible to study the methylation pattern and other covalent modifications of the core histones at defined genomic loci in postmortem brain. Here, we present a simplified native chromatin immunoprecipitation (NChIP) protocol for frozen (never-fixed) human brain specimens. Starting with micrococcal nuclease digestion of brain homogenates, NChIP followed by qPCR can be completed within three days. The methodology presented here should be useful to elucidate epigenetic mechanisms of gene expression in normal and diseased human brain.

Until recently, expression studies on human brain were limited to quantification of RNA or protein. With the chromatin immunoprecipitation techniques described in this paper, it will be possible to map histone methylation and other epigenetic regulators of gene expression in postmortem brain.

Un saggio cromatina per tessuto cerebrale umano

Chronic neuropsychiatric illnesses such as schizophrenia, bipolar disease and autism are thought to result from a combination of genetic and environmental ...

Ricerca

Biologia

Single Read and Paired End mRNA-Seq Illumina Libraries from 10 Nanograms Total RNA

Whole transcriptome sequencing by mRNA-Seq is now used extensively to perform global gene expression, mutation, allele-specific expression and other genome-wide analyses. mRNA-Seq even opens the gate for gene expression analysis of non-sequenced genomes. mRNA-Seq offers high sensitivity, a large dynamic range and allows measurement of transcript copy numbers in a sample. Illumina&#x2019;s genome analyzer performs sequencing of a large number (&gt; 107) of relatively short sequence reads (&lt; 150 bp).The &quot;paired end&quot; approach, wherein a single long read is sequenced at both its ends, allows for tracking alternate splice junctions, insertions and deletions, and is useful for de novo transcriptome assembly.
One of the major challenges faced by researchers is a limited amount of starting material. For example, in experiments where cells are harvested by laser micro-dissection, available starting total RNA may measure in nanograms. Preparation of mRNA-Seq libraries from such samples have been described1, 2 but involves significant PCR amplification that may introduce bias. Other RNA-Seq library construction procedures with minimal PCR amplification have been published3, 4 but require microgram amounts of starting total RNA.
Here we describe a protocol for the Illumina Genome Analyzer II platform for mRNA-Seq sequencing for library preparation that avoids significant PCR amplification and requires only 10 nanograms of total RNA. While this protocol has been described previously and validated for single-end sequencing5, where it was shown to produce directional libraries without introducing significant amplification bias, here we validate it further for use as a paired end protocol. We selectively amplify polyadenylated messenger RNAs from starting total RNA using the T7 based Eberwine linear amplification method, coined &quot;T7LA&quot; (T7 linear amplification). The amplified poly-A mRNAs are fragmented, reverse transcribed and adapter ligated to produce the final sequencing library. For both single read and paired end runs, sequences are mapped to the human transcriptome6 and normalized so that data from multiple runs can be compared. We report the gene expression measurement in units of transcripts per million (TPM), which is a superior measure to RPKM when comparing samples7.

Here we describe a method for preparation of both single read and paired end Illumina mRNA-Seq sequencing libraries for gene expression analysis based on T7 linear RNA amplification. This protocol requires only 10 nanograms of starting total RNA and generates highly consistent libraries representing whole transcripts.

Leggi singolo e accoppiamento finale mRNA-Seq Biblioteche Illumina da 10 nanogrammi di RNA totale

Whole transcriptome sequencing by mRNA-Seq is now used extensively to perform global gene expression, mutation, allele-specific expression and other ...

Chromatin Immunoprecipitation (ChIP) using Drosophila tissue

Epigenetics remains a rapidly developing field that studies how the chromatin state contributes to differential gene expression in distinct cell types at different developmental stages. Epigenetic regulation contributes to a broad spectrum of biological processes, including cellular differentiation during embryonic development and homeostasis in adulthood. A critical strategy in epigenetic studies is to examine how various histone modifications and chromatin factors regulate gene expression. To address this, Chromatin Immunoprecipitation (ChIP) is used widely to obtain a snapshot of the association of particular factors with DNA in the cells of interest.
ChIP technique commonly uses cultured cells as starting material, which can be obtained in abundance and homogeneity to generate reproducible data. However, there are several caveats: First, the environment to grow cells in Petri dish is different from that in vivo, thus may not reflect the endogenous chromatin state of cells in a living organism. Second, not all types of cells can be cultured ex vivo. There are only a limited number of cell lines, from which people can obtain enough material for ChIP assay.
Here we describe a method to do ChIP experiment using Drosophila tissues. The starting material is dissected tissue from a living animal, thus can accurately reflect the endogenous chromatin state. The adaptability of this method with many different types of tissue will allow researchers to address a lot more biologically relevant questions regarding epigenetic regulation in vivo1, 2. Combining this method with high-throughput sequencing (ChIP-seq) will further allow researchers to obtain an epigenomic landscape.

Chromatin Immunoprecipitation ChIP using Drosophila tissue

Recently high-throughput sequencing technology has greatly increased sensitivity of Chromatin Immunoprecipitation (ChIP) experiment and prompted its application using purified cells or dissected tissue. Here we delineate a method to use ChIP technique with Drosophila tissue, which can address the endogenous chromatin state in a well-characterized biological system.

Immunoprecipitazione della cromatina (ChIP) con Drosophila Tessuto

Epigenetics remains a rapidly developing field that studies how the chromatin state contributes to differential gene expression in distinct cell types at ...

Chromatin Isolation by RNA Purification (ChIRP)

Long noncoding RNAs are key regulators of chromatin states for important biological processes such as dosage compensation, imprinting, and developmental gene expression 1,2,3,4,5,6,7. The recent discovery of thousands of lncRNAs in association with specific chromatin modification complexes, such as Polycomb Repressive Complex 2 (PRC2) that mediates histone H3 lysine 27 trimethylation (H3K27me3), suggests broad roles for numerous lncRNAs in managing chromatin states in a gene-specific fashion 8,9. While some lncRNAs are thought to work in cis on neighboring genes, other lncRNAs work in trans to regulate distantly located genes. For instance, Drosophila lncRNAs roX1 and roX2 bind numerous regions on the X chromosome of male cells, and are critical for dosage compensation 10,11. However, the exact locations of their binding sites are not known at high resolution. Similarly, human lncRNA HOTAIR can affect PRC2 occupancy on hundreds of genes genome-wide 3,12,13, but how specificity is achieved is unclear. LncRNAs can also serve as modular scaffolds to recruit the assembly of multiple protein complexes. The classic trans-acting RNA scaffold is the TERC RNA that serves as the template and scaffold for the telomerase complex 14; HOTAIR can also serve as a scaffold for PRC2 and a H3K4 demethylase complex 13.
Prior studies mapping RNA occupancy at chromatin have revealed substantial insights 15,16, but only at a single gene locus at a time. The occupancy sites of most lncRNAs are not known, and the roles of lncRNAs in chromatin regulation have been mostly inferred from the indirect effects of lncRNA perturbation. Just as chromatin immunoprecipitation followed by microarray or deep sequencing (ChIP-chip or ChIP-seq, respectively) has greatly improved our understanding of protein-DNA interactions on a genomic scale, here we illustrate a recently published strategy to map long RNA occupancy genome-wide at high resolution 17. This method, Chromatin Isolation by RNA Purification (ChIRP) (Figure 1), is based on affinity capture of target lncRNA:chromatin complex by tiling antisense-oligos, which then generates a map of genomic binding sites at a resolution of several hundred bases with high sensitivity and low background. ChIRP is applicable to many lncRNAs because the design of affinity-probes is straightforward given the RNA sequence and requires no knowledge of the RNA's structure or functional domains.

Chromatin Isolation by RNA Purification ChIRP

ChIRP is a novel and rapid technique to map genomic binding sites of long noncoding RNAs (lncRNAs). The method takes advantage of the specificity of anti-sense tiling oligonucleotides to allow the enumeration of lncRNA-bound genomic sites.

Isolamento di cromatina Purificazione RNA (disturbo)

Long noncoding RNAs are key regulators of chromatin states for important biological processes such as dosage compensation, imprinting, and developmental ...

Generation of High Quality Chromatin Immunoprecipitation DNA Template for High-throughput Sequencing (ChIP-seq)

ChIP-sequencing (ChIP-seq) methods directly offer whole-genome coverage, where combining chromatin immunoprecipitation (ChIP) and massively parallel sequencing can be utilized to identify the repertoire of mammalian DNA sequences bound by transcription factors in vivo. "Next-generation" genome sequencing technologies provide 1-2 orders of magnitude increase in the amount of sequence that can be cost-effectively generated over older technologies thus allowing for ChIP-seq methods to directly provide whole-genome coverage for effective profiling of mammalian protein-DNA interactions.
For successful ChIP-seq approaches, one must generate high quality ChIP DNA template to obtain the best sequencing outcomes. The description is based around experience with the protein product of the gene most strongly implicated in the pathogenesis of type 2 diabetes, namely the transcription factor transcription factor 7-like 2 (TCF7L2). This factor has also been implicated in various cancers.
Outlined is how to generate high quality ChIP DNA template derived from the colorectal carcinoma cell line, HCT116, in order to build a high-resolution map through sequencing to determine the genes bound by TCF7L2, giving further insight in to its key role in the pathogenesis of complex traits.

Generation of High Quality Chromatin Immunoprecipitation DNA Template for High-throughput Sequencing ChIP-seq

The combination of chromatin immunoprecipitation and ultra-high-throughput sequencing (ChIP-seq) can identify and map protein-DNA interactions in a given tissue or cell line. Outlined is how to generate a high quality ChIP template for subsequent sequencing, using experience with the transcription factor TCF7L2 as an example.

Generazione di immunoprecipitazione della cromatina Template DNA di alta qualità per high-throughput sequencing (ChIP-Seq)

ChIP-sequencing (ChIP-seq) methods directly  offer whole-genome coverage, where combining chromatin immunoprecipitation  (ChIP) and massively parallel ...

An Efficient Method for Quantitative, Single-cell Analysis of Chromatin Modification and Nuclear Architecture in Whole-mount Ovules in Arabidopsis

In flowering plants, the somatic-to-reproductive cell fate transition is marked by the specification of spore mother cells (SMCs) in floral organs of the adult plant. The female SMC (megaspore mother cell, MMC) differentiates in the ovule primordium and undergoes meiosis. The selected haploid megaspore then undergoes mitosis to form the multicellular female gametophyte, which will give rise to the gametes, the egg cell and central cell, together with accessory cells. The limited accessibility of the MMC, meiocyte and female gametophyte inside the ovule is technically challenging for cytological and cytogenetic analyses at single cell level. Particularly, direct or indirect immunodetection of cellular or nuclear epitopes is impaired by poor penetration of the reagents inside the plant cell and single-cell imaging is demised by the lack of optical clarity in whole-mount tissues.
Thus, we developed an efficient method to analyze the nuclear organization and chromatin modification at high resolution of single cell in whole-mount embedded Arabidopsis ovules. It is based on dissection and embedding of fixed ovules in a thin layer of acrylamide gel on a microscopic slide. The embedded ovules are subjected to chemical and enzymatic treatments aiming at improving tissue clarity and permeability to the immunostaining reagents. Those treatments preserve cellular and chromatin organization, DNA and protein epitopes. The samples can be used for different downstream cytological analyses, including chromatin immunostaining, fluorescence in situ hybridization (FISH), and DNA staining for heterochromatin analysis. Confocal laser scanning microscopy (CLSM) imaging, with high resolution, followed by 3D reconstruction allows for quantitative measurements at single-cell resolution.

An Efficient Method for Quantitative, Single-cell Analysis of Chromatin Modification and Nuclear Architecture in Whole-mount Ovules in Arabidopsis

We provide here an efficient and reliable protocol for immunostaining, Fluorescence in&#160;situ Hybridization, DNA staining followed by quantitative, high-resolution imaging in whole-mount Arabidopsis thaliana ovules. This method was successfully used to analyze chromatin modifications and nuclear architecture.

Un metodo efficiente per quantitativa, analisi singola cella della cromatina Cambiamento e architettura nucleare in tutto il montaggio Ovuli in<em&gt; Arabidopsis</em

In flowering plants, the somatic-to-reproductive cell fate transition is marked by the specification of spore mother cells (SMCs) in floral organs of the ...

Generation and Purification of Human INO80 Chromatin Remodeling Complexes and Subcomplexes

INO80 chromatin remodeling complexes regulate nucleosome dynamics and DNA accessibility by catalyzing ATP-dependent nucleosome remodeling. Human INO80 complexes consist of 14 protein subunits including Ino80, a SNF2-like ATPase, which serves both as the catalytic subunit and the scaffold for assembly of the complexes. Functions of the other subunits and the mechanisms by which they contribute to the INO80 complex&#39;s chromatin remodeling activity remain poorly understood, in part due to the challenge of generating INO80 subassemblies in human cells or heterologous expression systems. This JOVE protocol describes a procedure that allows purification of human INO80 chromatin remodeling subcomplexes that are lacking a subunit or a subset of subunits. N-terminally FLAG epitope tagged Ino80 cDNA are stably introduced into human embryonic kidney (HEK) 293 cell lines using Flp-mediated recombination. In the event that a subset of subunits of the INO80 complex is to be deleted, one expresses instead mutant Ino80 proteins that lack the platform needed for assembly of those subunits. In the event an individual subunit is to be depleted, one transfects siRNAs targeting this subunit into an HEK 293 cell line stably expressing FLAG tagged Ino80 ATPase. Nuclear extracts are prepared, and FLAG immunoprecipitation is performed to enrich protein fractions containing Ino80 derivatives. The compositions of purified INO80 subcomplexes can then be analyzed using methods such as immunoblotting, silver staining, and mass spectrometry. The INO80 and INO80 subcomplexes generated according to this protocol can be further analyzed using various biochemical assays, which are described in the accompanying JOVE protocol. The methods described here can be adapted for studies of the structural and functional properties of any mammalian multi-subunit chromatin remodeling and modifying complexes.

This protocol describes a procedure for generating and purifying wild type and mutant versions of the human INO80 chromatin remodeling complex. Epitope tagged versions of INO80 subunits are stably expressed in HEK293 cells, and complete complexes and complexes lacking specific sets of subunits are purified by immunoaffinity chromatography.

Generazione e Purificazione di umani INO80 rimodellamento della cromatina Complessi e Sottocomplessi

INO80 chromatin remodeling complexes regulate nucleosome dynamics and DNA accessibility by catalyzing ATP-dependent nucleosome remodeling. Human INO80 ...

Chromatin Immunoprecipitation Assay for the Identification of Arabidopsis Protein-DNA Interactions In Vivo

Intricate gene regulatory networks orchestrate biological processes and developmental transitions in plants. Selective transcriptional activation and silencing of genes mediate the response of plants to environmental signals and developmental cues. Therefore, insights into the mechanisms that control plant gene expression are essential to gain a deep understanding of how biological processes are regulated in plants. The chromatin immunoprecipitation (ChIP) technique described here is a procedure to identify the DNA-binding sites of proteins in genes or genomic regions of the model species Arabidopsis thaliana. The interactions with DNA of proteins of interest such as transcription factors, chromatin proteins or posttranslationally modified versions of histones can be efficiently analyzed with the ChIP protocol. This method is based on the fixation of protein-DNA interactions in vivo, random fragmentation of chromatin, immunoprecipitation of protein-DNA complexes with specific antibodies, and quantification of the DNA associated with the protein of interest by PCR techniques. The use of this methodology in Arabidopsis has contributed significantly to unveil transcriptional regulatory mechanisms that control a variety of plant biological processes. This approach allowed the identification of the binding sites of the Arabidopsis chromatin protein EBS to regulatory regions of the master gene of flowering FT. The impact of this protein in the accumulation of particular histone marks in the genomic region of FT was also revealed through ChIP analysis.

Chromatin Immunoprecipitation Assay for the Identification of Arabidopsis Protein-DNA Interactions In Vivo

Chromatin immunoprecipitation is a powerful technique for the identification of DNA binding sites of Arabidopsis proteins in vivo. This procedure includes chromatin cross-linking and fragmentation, immunoprecipitation with selective antibodies against the protein of interest, and qPCR analysis of bound DNA. We describe a simple ChIP assay optimized for Arabidopsis plants.

Immunoprecipitazione della cromatina Saggio per la identificazione di Arabidopsis interazioni proteina-DNA<em&gt; In Vivo</em

Intricate gene regulatory networks orchestrate biological processes and developmental transitions in plants. Selective transcriptional activation and ...

Isolation of Specific Genomic Regions and Identification of Associated Molecules by enChIP

The identification of molecules associated with specific genomic regions of interest is required to understand the mechanisms of regulation of the functions of these regions. To enable the non-biased identification of molecules interacting with a specific genomic region of interest, we recently developed the engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP) technique. Here, we describe how to use enChIP to isolate specific genomic regions and identify the associated proteins and RNAs. First, a genomic region of interest is tagged with a transcription activator-like (TAL) protein or a clustered regularly interspaced short palindromic repeats (CRISPR) complex consisting of a catalytically inactive form of Cas9 and a guide RNA. Subsequently, the chromatin is crosslinked and fragmented by sonication. The tagged locus is then immunoprecipitated and the crosslinking is reversed. Finally, the proteins or RNAs that are associated with the isolated chromatin are subjected to mass spectrometric or RNA sequencing analyses, respectively. This approach allows the successful identification of proteins and RNAs associated with a genomic region of interest.

The identification of molecules associated with specific genomic regions of interest is required to understand the mechanisms of regulation of the functions of these regions. This protocol describes procedures to perform engineered DNA-binding molecule-mediated chromatin imunoprecipitation (enChIP) for identification of proteins and RNAs associated with a specific genomic region.

L&#39;isolamento di regioni genomiche specifici e l&#39;identificazione di molecole da Associated enChIP

The identification of molecules associated with specific genomic regions of interest is required to understand the mechanisms of regulation of the ...

Genome-wide Mapping of Drug-DNA Interactions in Cells with COSMIC (Crosslinking of Small Molecules to Isolate Chromatin)

The genome is the target of some of the most effective chemotherapeutics, but most of these drugs lack DNA sequence specificity, which leads to dose-limiting toxicity and many adverse side effects. Targeting the genome with sequence-specific small molecules may enable molecules with increased therapeutic index and fewer off-target effects. N-methylpyrrole/N-methylimidazole polyamides are molecules that can be rationally designed to target specific DNA sequences with exquisite precision. And unlike most natural transcription factors, polyamides can bind to methylated and chromatinized DNA without a loss in affinity. The sequence specificity of polyamides has been extensively studied in vitro with cognate site identification (CSI) and with traditional biochemical and biophysical approaches, but the study of polyamide binding to genomic targets in cells remains elusive. Here we report a method, the crosslinking of small molecules to isolate chromatin (COSMIC), that identifies polyamide binding sites across the genome. COSMIC is similar to chromatin immunoprecipitation (ChIP), but differs in two important ways: (1) a photocrosslinker is employed to enable selective, temporally-controlled capture of polyamide binding events, and (2) the biotin affinity handle is used to purify polyamide&#8211;DNA conjugates under semi-denaturing conditions to decrease DNA that is non-covalently bound. COSMIC is a general strategy that can be used to reveal the genome-wide binding events of polyamides and other genome-targeting chemotherapeutic agents.

Genome-wide Mapping of Drug-DNA Interactions in Cells with COSMIC Crosslinking of Small Molecules to Isolate Chromatin

Identifying the direct targets of genome-targeting molecules remains a major challenge. To understand how DNA-binding molecules engage the genome, we developed a method that relies on crosslinking of small molecules to isolate chromatin (COSMIC).