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Small RNA sequencing of circulating exosomes from SD rats

ABSTRACT: After total RNA was extracted by Trizol reagent kit (Invitrogen, Carlsbad, CA,USA), the RNA molecules in a size range of 18–30nt were enriched by polyacrylamide gelelectrophoresis(PAGE). Then the 3’ adapters were added and the 36-48nt RNAs were enriched. The 5’ adapters were then ligated to the RNAs as well. The ligation products were reverse transcribed by PCR amplification and the 140-160bp size PCR products were enriched to generate a cDNA library and sequenced using Illumina HiSeq Xten by Gene Denovo Biotechnology Co. (Guangzhou, China).

ORGANISM(S): Rattus norvegicus

PROVIDER: GSE298553 | GEO | 2026/04/01

REPOSITORIES: GEO

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Project description:This study describes the application of the LongSAGE methodology to study the gene expression profile in Leishmania infantum chagasi. A tag library was created using the LongSAGE method and consisted of 14,208 tags of 17 bases. Of these, 8,427 (59.3%) were distinct. BLAST research of the 1,645 most abundant tags showed that 12.8% of them identified the coding sequences of genes, while 82% (1,349/1,645) identified one or more genomic sequences that did not correspond with open reading frames. Only 5.2% (84/1,645) of the tags were not aligned to any position in the L. infantum genome. The UTR size of Leishmania and the lack of CATG sites in some transcripts were decisive for the generation of tags in these regions. LongSAGE revealed the gene expression profile in promastigotes of L. i. chagasi. Additional analysis will allow a better understanding of the expression profile and discovering the key genes in this life cycle. The I-SAGETM Long kit (Invitrogen) was used to construct the L. i. chagasi library according to the manufacturer's recommendations. The following were included among the main steps: polyadenylates mRNAs were captured by oligo (dT) linked to magnetic beads and used for cDNA synthesis. The cDNA was digested with 60 U of NlaIII (anchoring enzyme) and the 3' ends of the cDNAs were isolated using the beads. The resulting cDNA 3' was divided into two equal portions and connected to two LongSAGE adapters, A and B. The long tags were released by the enzyme MmeI and linked to form ~130 bp ditags. Dilutions of 1:40 of the product were amplified in 27 PCR cycles (300 reactions in total). The precipitated PCR products were separated on 12% polyacrylamide gel and the 130 bp bands were cut out and the precipitated DNA was digested with NlaIII. The digestion products were separated on 12% polyacrylamide gel and ~34 bp ditags were cut out and linked to form concatemers. The concatemers were separated on 6% polyacrylamide gel and the fractions of 250-500 and 500-800pb were isolated and cloned into pZErO-1 vector digested with SphI. Vectors with concatemers were cloned in E. coli DH10b by electroporation and the isolated recombinant vectors were used as template for sequencing in the ABI Prism 3100 (Applied Biosystems).

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Small RNA sequencing of circulating exosomes from SD rats

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