Home › Genetics / Genomics › 3' End Sequencing Library Preparation with A-seq2
Genetics / Genomics JoVE (Open Access) Citable · DOI

3' End Sequencing Library Preparation with A-seq2

DOI: 10.3791/56129-v
What you'll learn
  • ✓ Prepare 3' end sequencing libraries using the A-seq2 protocol
  • ✓ Map pre-mRNA 3' end processing sites from cell-derived mRNA
  • ✓ Perform adapter ligation, reverse transcription, and library amplification
  • ✓ Analyze sequencing data to identify polyadenylation sites
Protocol

This protocol describes a method for mapping pre-mRNA 3' end processing sites.

Difficulty
advanced
Total time
~3-4 days (mRNA isolation through computational analysis)
Biosafety
BSL-1

Steps

1
Isolate mRNA from cultured cells

Extract and purify mRNA from cell samples using standard isolation techniques. This serves as the starting material for 3' end mapping.

▶ 00:46
2
Phosphorylate 5' ends and block 3' ends

Treat mRNA with kinase to phosphorylate 5' ends, perform DNase digestion, and apply 3' end blocking chemistry to prepare RNA for adapter ligation.

▶ 03:24
3
Ligate reverse adapters and perform reverse transcription

Anneal reverse 3' adapters to the 5' phosphorylated end of RNA fragments and synthesize complementary cDNA using reverse transcriptase.

▶ 04:30
4
Digest uracil residues and ligate 5' adapters

Treat cDNA with uracil DNA glycosylase to remove uracil-containing strands, then ligate 5' adapters to prepare for PCR amplification.

▶ 05:59
5
Amplify library and select by size

Perform pilot PCR to determine optimal cycle number, then conduct full-scale amplification of sequencing libraries and purify products by size selection.

▶ 07:44
6
Analyze sequencing data computationally

Process and analyze DNA sequence reads to identify and map pre-mRNA 3' end processing sites genome-wide.

▶ 09:21
💬 Comments coming soon