Project description:To identify full-length cap-to-poly(A) mRNA isoforms of CD20 and rule out reverse transcription artifacts which are common in cDNA-seq approaches, long-read Oxford Nanopore direct RNA sequencing was performed on the Raji cell line.

2023-12-08 | GSE243920 | GEO

Nanopore native RNA-seq for genome-wide identification of transcription units and transcriptional termination sites in the phytopathogen Dickeya dadantii 3937

Project description:In this study, based on Nanopore direct RNA-seq where native RNAs are sequenced directly as near full-length transcripts in the 3' to 5' direction, transcription units of the phytopathogen Dickeya dadantii 3937 were validated and transcriptional termination sites were determined. Briefly, D. dadantii cultures were grown in M63 medium supplemented with 0.2% glucose and 0.2% PGA, until the early exponential phase (A600nm = 0.2, condition 1), or the early stationary phase (A600nm = 1.8, condition 2). RNAs were extracted using a frozen acid-phenol method, as previously described (Hommais et al. 2008), and treated successively with Roche and Biolabs DNases. Two samples were prepared: 50 µg of RNAs from each condition were pulled into one sample (sample 1), whereas the other one contained 100 µg of RNAs from condition 2 (sample 2). Both samples were then supplied to Vertis Biotechnologie AG for Nanopore native RNA-seq: total RNA preparations were first examined by capillary electrophoresis. For sample 1, ribosomal RNA molecules were depleted using an in-house developed protocol (recovery rate = 84%), whereas no ribodepletion was performed for sample 2. 3' ends of RNA were then poly(A)-tailed using poly(A) polymerase, and the Direct RNA sequencing kit (SQK-RNA002) was used to prepare the library for 1D sequencing on the Oxford Nanopore sequencing device. The direct RNA libraries were sequenced on a MinION device (MIN-101B) using standard settings. Basecalling of the fast5 files was performed using Guppy (version 3.6.1) with the following settings: --flowcell FLO-MIN106 --kit SQK-RNA002 --cpu_threads_per_caller 12--compress_fastq --reverse_sequence true --trim_strategy rna. Reads smaller than 50 nt were removed. 466 393 and 556 850 reads were generated for sample 1 and 2, respectively.

2022-01-01 | E-MTAB-10482 | biostudies-arrayexpress

Project description:Immobilized Enzymes for Nanopore Direct RNA-Seq of Non-Poly(A) Tailed RNA

| PRJNA683132 | ENA

PAT-seq - a high-throughput polyadenylation assay for the Illumina platform

Project description:Polyadenylation - the cleavage of the nascent mRNA and the addition of a tract consisting of adenine bases in a non-template-dependent manner - is a post-transcriptional modification characteristic of the majority of eukaryotic transcripts. In this study we explore PAT-seq, an Illumina-based poly(A) tail assay, based on the low-throughput G-tailing approach. Briefly, our approach is based on G-tailing poly(A) selected mRNA followed by fragmentation and reverse transcription using a mixture of primers targeting the poly(A)-tail/G-tag junction and the polyadenylation site and the body of the transcripts. The resulting cDNA fragments are sequenced using standard Illumina RNA-seq protocol.

2014-04-05 | E-MTAB-2456 | biostudies-arrayexpress

Total poly(A)+ RNA sequencing

Project description:Total poly(A)+ RNA sequencing

| PRJNA627618 | ENA

Quartz-Seq: a simple and highly quantitative method for single-cell RNA-Seq

Project description:We report novel single-cell RNA-Seq, called Quartz-Seq. Quartz-Seq was simplified method compared with previous methods based on poly-A tailing reaction.

2013-04-04 | GSE42268 | GEO

Nanopore RNA sequencing in S. cerevisiae strains defective for decapping and deadenylation

Project description:RNA turnover in eukaryotes is triggered by decapping, believed to be activated mostly after deadenylation. To investigate simultaneous changes in RNA levels and poly(A) tail size we performed Nanopore RNA sequencing on total RNA extracted from S. cerevisiae strains in which the decapping enzyme Dcp2, or the deadenylation factors Ccr4 and Pop2 were depleted through an inducible degron system. Two fully independent replicate experiments were performed for wild type and the two mutant conditions.

2022-10-01 | GSE211782 | GEO

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