A549 cells before/after NME2 induction
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
SUBMITTER: Vinod Yadav
PROVIDER: E-GEOD-40433 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
Publications
Promoter-proximal transcription factor binding is transcriptionally active when coupled with nucleosome repositioning in immediate vicinity.
Yadav Vinod Kumar VK Thakur Ram Krishna RK Eckloff Bruce B Baral Aradhita A Singh Ankita A Halder Rashi R Kumar Akinchan A Alam Mohammad Parwez MP Kundu Tapas K TK Pandita Raj R Pandita Tej K TK Wieben Eric D ED Chowdhury Shantanu S
Nucleic acids research 20140731 15
Previous studies have analyzed patterns of transcription, transcription factor (TF) binding or mapped nucleosome occupancy across the genome. These suggest that the three aspects are genetically connected but the cause and effect relationships are still unknown. For example, physiologic TF binding studies involve many TFs, consequently, it is difficult to assign nucleosome reorganization to the binding site occupancy of any particular TF. Therefore, several aspects remain unclear: does TF bindin ...[more]
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Briefly, RNA was isolated from lyophilized cells by vortexing with glass beads (3.0 mm, acid-washed and RNase-free) for 15 min in 15 mL of TRIZOL extraction buffer (Invitrogen, Carlsbad, CA, USA). The mixture was incubated for 15 min at room temperature and total RNA was isolated according to the manufacturer's instructions. Total RNA was used directly for SAGE library construction as described by Velculescu et al. (1995) using the I-Long SAGE kit (Invitrogen). The tagging enzyme for cDNA digestion was NlaIII and 29 PCR cycles were performed to amplify the ditags during library construction. Colonies were screened by PCR (M13F and M13R primers) to assess the average clone insert size and the percentage of recombinants. Clones from the libraries were sequenced by BigDye primer cycle sequencing on an ABI PRISM 3700 DNA analy zer. Sequence chromatograms were processed using PHRED (Ewing et al., 1998), and vector sequence was detected using Cross_match (Gordon et al., 1998). Fourteen-base-pair tags were extracted from the vector-clipped sequence, and an overall quality score for each tag was derived based on the cumulative PHRED score. Duplicate ditags and linker sequences were removed as described previously (Steen et al., 2002). Only tags with a predicted accuracy of 99% were used, and statistical differences between tag abundance in different libraries were determined as described (Audic and Claverie, 1997). The libraries yielded 23,904 tags from cells grown in media without macrophages for 6hr and 38,5444 tags from cells grown in media with macrophages for 6hr. An overview of the abundance classes for both SAGE libraries is presented in Table S1 in the supplementary material, with both the number of different tag sequences and the total number of tags present in each class for the cells from th e 6h infection and the control. All libraries were normalized to 23,904 to allow direct comparisons, and the tags that appeared less than once in any given library were removed. The EST database available for strain H99 at the University of Oklahoma's Advanced Center for Genome Technology http://www.genome.ou.edu/cneo.html) was used for the preliminary assignment of tags to genes. When an EST sequence could not be identified for a particular tag, the genomic sequence for H99 at the Duke University Center for Genome Technology (http://cgt.genetics.duke.edu/data/index.html) and the Broad Institute (http://cneo.genetics.duke.edu/) was used to identify contigs with unambiguous tag assignments. Note that a limitation of the SAGE approach is that some transcripts are not detected because of low abundance and/or the absence of an NlaIII site for transcript processing. The control (fungal cells alone) and cryptococcal-macrophage interaction SAGE datasets were deposited to the NCBI under accession number GSM986206 and GSM986207 respectively.
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