Reverse Transcriptase Template Switching: A SMART™ Approach for Full-Length cDNA Library Construction

Yy, Zhu; Em, Machleder; Chenchik, Alex; R, Li; Pd, Siebert

doi:10.2144/01304pf02

Cited by 681 publications

(578 citation statements)

References 22 publications

Supporting

Mentioning

558

Contrasting

Unclassified

Order By: Relevance

“…In this screening, one positive control of H4 protein, H4K5acK8ac, and four negative controls of H4 protein (unmodified H4, H4K5ac, H4K8ac, and H4K8acK12ac) were used. cDNA were prepared from total RNA isolated from the hit hybridoma clones 1A9D7 and 2A7D9 and sequenced essentially as previously described [57]. …”

Section: Methodsmentioning

confidence: 99%

JQ1 affects BRD2-dependent and independent transcription regulation without disrupting H4-hyperacetylated chromatin states

Handoko¹,

Kaczkowski²,

Hon³

et al. 2018

Epigenetics

View full text Add to dashboard Cite

The bromodomain and extra-terminal domain (BET) proteins are promising drug targets for cancer and immune diseases. However, BET inhibition effects have been studied more in the context of bromodomain-containing protein 4 (BRD4) than BRD2, and the BET protein association to histone H4-hyperacetylated chromatin is not understood at the genome-wide level. Here, we report transcription start site (TSS)-resolution integrative analyses of ChIP-seq and transcriptome profiles in human non-small cell lung cancer (NSCLC) cell line H23. We show that di-acetylation at K5 and K8 of histone H4 (H4K5acK8ac) co-localizes with H3K27ac and BRD2 in the majority of active enhancers and promoters, where BRD2 has a stronger association with H4K5acK8ac than H3K27ac. Although BET inhibition by JQ1 led to complete reduction of BRD2 binding to chromatin, only local changes of H4K5acK8ac levels were observed, suggesting that recruitment of BRD2 does not influence global histone H4 hyperacetylation levels. This finding supports a model in which recruitment of BET proteins via histone H4 hyperacetylation is predominant over hyperacetylation of histone H4 by BET protein-associated acetyltransferases. In addition, we found that a remarkable number of BRD2-bound genes, including MYC and its downstream target genes, were transcriptionally upregulated upon JQ1 treatment. Using BRD2-enriched sites and transcriptional activity analysis, we identified candidate transcription factors potentially involved in the JQ1 response in BRD2-dependent and -independent manner.

show abstract

Section: Methodsmentioning

confidence: 99%

JQ1 affects BRD2-dependent and independent transcription regulation without disrupting H4-hyperacetylated chromatin states

Handoko¹,

Kaczkowski²,

Hon³

et al. 2018

Epigenetics

View full text Add to dashboard Cite

show abstract

“…For each library except for testis and liver (hibernating animal only), we pooled mRNA samples isolated from hibernating and summer active bears. During reverse transcription, adaptors containing the asymmetrical restriction sites for SfiI were incorporated into the first strand of the cDNA using a SMART template-switching mechanism at the 5Ј-end of the transcript (47). To decrease redundancy, all libraries were normalized by hybridization of the single-strand cDNA with the same pool of mRNA that was used for first-strand synthesis (9).…”

Section: Methodsmentioning

confidence: 99%

Elevated expression of protein biosynthesis genes in liver and muscle of hibernating black bears (Ursus americanus)

Fedorov

Goropashnaya

Tøien

et al. 2009

Physiological Genomics

125

View full text Add to dashboard Cite

We conducted a large-scale gene expression screen using the 3,200 cDNA probe microarray developed specifically for Ursus americanus to detect expression differences in liver and skeletal muscle that occur during winter hibernation compared with animals sampled during summer. The expression of 12 genes, including RNA binding protein motif 3 (Rbm3), that are mostly involved in protein biosynthesis, was induced during hibernation in both liver and muscle. The Gene Ontology and Gene Set Enrichment analysis consistently showed a highly significant enrichment of the protein biosynthesis category by overexpressed genes in both liver and skeletal muscle during hibernation. Coordinated induction in transcriptional level of genes involved in protein biosynthesis is a distinctive feature of the transcriptome in hibernating black bears. This finding implies induction of translation and suggests an adaptive mechanism that contributes to a unique ability to reduce muscle atrophy over prolonged periods of immobility during hibernation. Comparing expression profiles in bears to small mammalian hibernators shows a general trend during hibernation of transcriptional changes that include induction of genes involved in lipid metabolism and carbohydrate synthesis as well as depression of genes involved in the urea cycle and detoxification function in liver. gene expression; hibernation; translation; RNA binding protein motif 3 MAMMALIAN HIBERNATION IS a physiological and behavioral adaptation involving a coordinated suppression of heat production and body temperature wherein whole body metabolism and energy demand are significantly reduced over several days to several months. During hibernation, heart and respiration rates, blood flow, and oxygen consumption decrease dramatically to Ͻ10% of normal or basal rates (2, 4). These physiological changes do not represent a loss of homeostasis but instead are precisely controlled and spontaneously reversible, and they allow individual animals to survive highly seasonal or unpredictable environments where food availability becomes lacking (7, 12). The molecular and genetic basis of hibernation in small mammals has only recently begun to be described, and little is known about its evolutionary history. Hibernating species have been found in diverse families among seven orders of mammals (26), however, and the interspersed phylogenetic distribution of hibernating and nonhibernating species has led to the hypothesis that rather than requiring the creation of novel gene products, hibernation results from the differential expression of genes that exist widely among mammals (35).Microarray technology provides powerful means for the unbiased detection of differences in expression of thousands of genes in a single hybridization experiment (14). In contrast to single-gene expression analysis, the genome-wide approach also allows for identification of coordinated transcriptional changes in functional groups of regulatory genes within metabolic and signaling pathways. Recent studies of differential gen...

show abstract

“…During formation of the library, base pairs were removed from the 5' end of the cDNA to create blunt ends for ligation of adaptor sequences (Xu & Faisal 2008). Therefore, in several sequences where base pairs were removed from the 5 0 translated region, the virtual protein sequences were incomplete at the N-terminus (Zhu et al 2001). An incomplete N-terminus also means that the correct reading frame of the cDNA sequence is not known and hence it is necessary to theoretically translate the cDNA in all three positive reading frames.…”

Section: Protein Identification and Sequence Analysismentioning

confidence: 99%

Byssal proteins of the freshwater zebra mussel,Dreissena polymorpha

2012

View full text Add to dashboard Cite

The freshwater zebra mussel (Dreissena polymorpha) is a notorious biofouling organism. It adheres to a variety of substrata underwater by means of a proteinaceous structure called the byssus, which consists of a number of threads with adhesive plaques at the tips. The byssal proteins are difficult to characterize due to extensive cross-linking of 3,4-dihydroxyphenylalanine (DOPA), which renders the mature structure largely resistant to protein extraction and immunolocalization. By inducing secretion of fresh threads and plaques in which cross-linking is minimized, three novel zebra mussel byssal proteins were identified following extraction and separation by gel electrophoresis. Peptide fragment fingerprinting was used to match tryptic digests of several gel bands against a cDNA library of genes expressed uniquely in the mussel foot, the organ which secretes the byssus. This allowed identification of a more complete sequence of Dpfp2 (D. polymorpha foot protein 2), a known DOPA-containing byssal protein, and a partial sequence of Dpfp5, a novel protein with several typical characteristics of mussel adhesive proteins.

show abstract

Reverse Transcriptase Template Switching: A SMART™ Approach for Full-Length cDNA Library Construction

Cited by 681 publications

References 22 publications

JQ1 affects BRD2-dependent and independent transcription regulation without disrupting H4-hyperacetylated chromatin states

JQ1 affects BRD2-dependent and independent transcription regulation without disrupting H4-hyperacetylated chromatin states

Elevated expression of protein biosynthesis genes in liver and muscle of hibernating black bears (Ursus americanus)

Byssal proteins of the freshwater zebra mussel,Dreissena polymorpha

Contact Info

Product

Resources

About