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Full-length poly(A) and mRNA sequencing (FLAM-seq)

Ivano Legnini, Jonathan Alles, Nikos Karaiskos, Salah Ayoub, Claudia Quedenau, Nikolaus Rajewsky
DOI: 10.21203/rs.2.10045/v1

Abstract

We developed FLAM-seq, a fast and simple method for generating cDNA libraries of full-length mRNAs, including the poly(A) tail. By combining a new strategy for cDNA preparation with PacBio long-read sequencing, FLAM-seq enables to generate hundreds of thousands of reads per sample in an easy and short procedure, with starting material ranging from 500 ng to 10 μg of total RNA. Besides the quantification of gene expression and the information about the mRNA isoform, the data generated with FLAM-seq allow accurate measurement of poly(A) tail length for thousands of genes in the sequenced samples. Here we describe the protocol step by step from the RNA preparation to the first steps of data analysis.

Keywords
RNA, long reads, full-length mRNA sequencing, poly(A) tails

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Preprint: Please note that this article has not completed peer review.

Full-length poly(A) and mRNA sequencing (FLAM-seq)

Ivano Legnini, Jonathan Alles, Nikos Karaiskos, Salah Ayoub, Claudia Quedenau, Nikolaus Rajewsky

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Abstract

We developed FLAM-seq, a fast and simple method for generating cDNA libraries of full-length mRNAs, including the poly(A) tail. By combining a new strategy for cDNA preparation with PacBio long-read sequencing, FLAM-seq enables to generate hundreds of thousands of reads per sample in an easy and short procedure, with starting material ranging from 500 ng to 10 μg of total RNA. Besides the quantification of gene expression and the information about the mRNA isoform, the data generated with FLAM-seq allow accurate measurement of poly(A) tail length for thousands of genes in the sequenced samples. Here we describe the protocol step by step from the RNA preparation to the first steps of data analysis.

Figures

Reagents

Equipment

Procedure

Troubleshooting

Learn more about our company.