| The nucleus is the eukryotic-specific,biggest and highly in-homogenous organelle with numerous sub-organelle and compartments.The chromatin is the existence of genetic and epigenetic information and is the largest single biological molecules.The around 2m-long chromatin is compacted within the nucleus of less than 10 um in diameters.Recently,the occurrence and the rapid progress of chromosome conformation capture(3C)and its series derivatives such as 4C,5C and Hi-C greatly promote the nucleus architecture study.It also revealed that some architecture proteins such as CTCF,Cohesin etc.play essential roles during the chromatin folding and interactions.Except the above mentioned proteins,very recently it also gradually discovered that some ncRNAs also involved in the chromatin interactions.By the interaction with DNA,Protein and even RNA itself,numerous ncRNAs such as XIST,Firre were found to mediate the chromatin interaction and regulated the formation of nuclear architecture.As the most part of the genome encoded noncoding RNA,we expected that there might be much more ncRNA involving the nuclear architecture regulation.But till now,there is not any systematic report about the ncRNAs participating in chromatin interactions.For the aim to screening the lncRNAs that might involve in the chromatin interactions genome-wide,we developed a Hi-C based high-throughput screening approached by the comparison of genome-wide chromatin interactome before and after RNase treatment.The high quality chromatin interactome or the Hi-C results is the key technique point of the whole research.Hi-C is a multi-steps,time-consuming molecular biology techniques.It involves many different kinds of reagents and machines.It is not robust,reproducibility is not so good and stable till now.Firstly,by the optimize the key steps of long-time,multi-steps complexed Hi-C experiments,such as crosslinking,pretreatment of digestion,inactivation of restriction enzyme and in situ ligation etc.,we developed an robust and stable Hi-C procedure.After the limited PCR amplifications of the primary Hi-C library,the two biological replicates of with and without RNA treatments of GM12878 samples were high-throughput sequenced.After the basic bioinformatics analysis,the quality and the reproducibility of the biological replicates were examined and it was found that in average,around 90% align-ability,72% pair-mate rate of raw data were obtained.Additionally,after remove of the self-circular ligation and danling-ends,more than 96% of the valid pairs were reached.The correlation analysis shown that each biological replicates is very strong correlated with each other in the term of both bin coverage and all bin pairs.All these results further proved that the modified Hi-C procedure is robust and very stable.After obtained the very high quality and high reproducible Hi-C results,we further compared the Hi-C results of with and without RNA treatment samples.The comparison shown that there are many interactions reduced or even disappeared after the RNase treatment,which is highly agreement with the observation that from the same quantity cell samples,around 1.58 fold Hi-C template or interactions of normal cells were reproducibly obtained comparing with that of the RNA treatment.After the overlapping and comparison of two biological replicates,we choose the top statistically 10000 different interactions for the further study.All the interactions sites from the top 10000 different interactions were aligned and merged with genome sites of the lncRNAs coding-gene.Finally 4081 lncRNAs were found that the nearby chromatin interactions were reduced after RNase treatment.The GO annotation revealed that the nearby genes of the 4081 lncRNAs mainly related to cell membrane,Pleckstrin homology-like domain,ammonium transporter,alternative splicing etc.These 4081 lncRNAs are the potential list that might involved in chromatin interactions and provide the good start list for the further molecular mechanism and functional study. |
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