Protocol for antibody-based m6A sequencing of human postmortem brain tissues

TL;DR

This paper provides a detailed protocol for detecting m6A RNA modifications in human postmortem brain tissues using antibody-based sequencing.

Contribution

An optimized protocol for m6A sequencing in postmortem brain tissue with step-by-step optimization data.

Findings

The protocol includes RNA extraction, shearing, immunoprecipitation, and library preparation steps.

Optimization data compares m6A profiles from total RNA and poly(A) RNA inputs.

Different commercial m6A antibodies and RNA/antibody ratios were evaluated for reliability.

Abstract

Here, we present an optimized N6-methyladenosine (m6A) immunoprecipitation sequencing for human postmortem brain tissue, building on previously established approaches. We describe steps for RNA extraction, RNA shearing, RNA immunoprecipitation, and library preparation. The protocol includes optimization data for each step, such as m6A profiles from total RNA versus poly(A) RNA as an input, a comparison of different commercial m6A antibodies, and immunoprecipitation with varying RNA/antibody ratios. This protocol allows reliable capture of m6A peaks from human postmortem brain tissue.

For complete details on the use and execution of this protocol, please refer to Mitsuhashi et al.1

•Steps for RNA extraction from human postmortem brain tissues•Instructions for mechanical RNA shearing prior to RNA immunoprecipitation•Procedures on RNA-IP with an m6A antibody followed by library preparation

Steps for RNA extraction from human postmortem brain tissues

Instructions for mechanical RNA shearing prior to RNA immunoprecipitation

Procedures on RNA-IP with an m6A antibody followed by library preparation

Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Here, we present an optimized N6-methyladenosine (m6A) immunoprecipitation sequencing for human postmortem brain tissue, building on previously established approaches. We describe steps for RNA extraction, RNA shearing, RNA immunoprecipitation, and library preparation. The protocol includes optimization data for each step, such as m6A profiles from total RNA versus poly(A) RNA as an input, a comparison of different commercial m6A antibodies, and immunoprecipitation with varying RNA/antibody ratios. This protocol allows reliable capture of m6A peaks from human postmortem brain tissue.

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