TruSeq Stranded mRNA

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	The protocol is optimized for 0.1–1 µg of total RNA.

Protocol tips
The protocol is optimized for 0.1–1 µg of total RNA.

Publication protocol

Purified mRNA samples from PBT003 cells were used for m6A-seq. RNA fragmentation was performed by
sonication at 10 ng μl-1 in 100 μl RNase-free water using Bioruptor Pico (Diagenode) with 30 s on / 30 s off cyclefor 30 cycles. m6A-immunoprecipitation (m6A IP) and library preparation were performed according to a published protocol. In detail, 2.5 μg affinity purified anti-m6A rabbit polyclonal antibody (Synaptic Systems; Catalog # 202003) and 20 μl Protein A beads (ThermoFisher; Catalog# 10002D) were used for each affinity pull-down. m6A antibody-bound RNAs were eluted with 100 μl elution buffer and recovered by RNA Clean and Concentrator-5 (Zymo), and subjected to RNA library preparation with TruSeq Stranded mRNA Library Prep Kit. Sequencing was carried out on Illumina HiSeq 4000 according to the manufacturer’s instructions.

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Manufacturer protocol

Download the product protocol from Illumina for TruSeq Stranded mRNA below.

Download manufacturer protocol

Videos

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