PAL-Seq

PAL-Seq

PAL-Seq measures poly(A)-tail length by incorporating fluorescent tags on biotinylated deoxyuridine triphosphate (dUTPs) and using signal intensity to quantify poly(A)-tail length. Similar to 3P-Seq RNA library preparation, a splint oligonucleotide containing a 3'-adapter sequence is ligated to the 3' end of polyadenylated RNA and partially digested with RNase T1.

To separate mRNA from total RNA, the sample is size-selected by gel purification and bound to streptavidin beads before phosphorylating the 5' ends for adapter ligation. Before cluster generation, the mRNA fragments are reverse-transcribed into cDNA, released from the beads, and purified by size selection through a gel. Sequencing primers are attached to the 3' end of the poly(A) sequence and extended using deoxythymidine triphosphate (dTTP) and biotinylated dUTP. To map the fragments, regions near the 5' end of the poly(A) tails are sequenced. Fluorescent-labeled streptavidin molecules are attached to the biotin-dUTPs, and their signal intensity is measured to quantify the length of the adenine homopolymers in each cluster.

Pros:

• Accurate measurement of poly(A)-tail length, regardless of length
• Avoids direct sequencing of the poly(A) tail

Cons:

• Technically complex to perform
• Efficiency-related issues may arise during the biotin-dUTP extension step
• Only captures 3' ends that consist purely of adenines

• PAL-Seq: Subtelny A. O., Eichhorn S. W., Chen G. R., Sive H. and Bartel D. P. (2014) Poly(A)-tail profiling reveals an embryonic switch in translational control. Nature 508: 66-71

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