Genome Res. 2019 Oct 24. doi:10.1101/gr.249656.119

Vahrenkamp JM, Szczotka K, Dodson MK, Jarboe EA, Soisson AP, Gertz J.

FFPEcap-seq: a method for sequencing capped RNAs in formalin-fixed paraffin-embedded samples.

Terminator, concatenation blocker, no manganese, indexes. Fails to cite our latest protocol.

Zhao B, Jin L, Wei J, Ma Z, Jiang W, Ma L, Jin Y.

IUBMB Life. 2012 Jul;64(7):612-6. doi:10.1002/iub.1026

A simple and fast method for profiling microRNA expression from low-input total RNA by microarray.

Poly-A tailing and template-switching. Required manganese

Genome Res. 1999 Jun;9(6):588-95

Korch C, Drabkin H.

Improved DNA sequencing accuracy and detection of heterozygous alleles using manganese citrate and different fluorescent dye terminators.

0.3 mM MnCl2 + 1:1 MnCit in absence of EDTA is optimal.

Tabor S, Richardson CC.

Proc Natl Acad Sci U S A. 1989 Jun;86(11):4076-80

Effect of manganese ions on the incorporation of dideoxynucleotides by bacteriophage T7 DNA polymerase and Escherichia coli DNA polymerase I.

Isocitrate used to buffer Mn2+ ions.

Oz-Gleenberg I, Herschhorn A, Hizi A.

Nucleic Acids Res. 2011 Feb;39(3):1042-53. doi: 10.1093/nar/gkq786

Reverse transcriptases can clamp together nucleic acids strands with two complementary bases at their 3'-termini for initiating DNA synthesis.

AT-rich cDNA tails are not triggering template-switching well. CC are the strongest. Mn₂⁺ potentiates template switching for MMLV. dGTP stabilises the clamp between the enzyme and the nucleic acids.

Nucleic Acids Res. 1999 Nov 1;27(21):e31.

Schmidt WM, Mueller MW.

CapSelect: a highly sensitive method for 5' CAP-dependent enrichment of full-length cDNA in PCR-mediated analysis of mRNAs.

Extra cytosine are more frequently added in presence of the 5′ cap. Increasing Mg2+ to 6 mM increases the frequency of addition of more than 1 C, but only moderately. Instead, when adding BSA in the reaction, supplementing it with 1 or 2 mM Mn2+ after 1h, 3-4 extra dC residues are added to most of the first strand cDNAs. Standard reaction: 0.75 μM oligo dT (or 0.25 μM gene-specific RT primer); 50 mM Tris-HCl (pH 8.3); 75 mM KCl; 3 mM MgCl2; 5 mM DTT; dNTPs 1mM each; 0.1 mg BSA; 20 U RNAse inhibitor (Roche), 200 U SuperScript II; 1h at 42 °C.

Manganese stimulates enzyme by destabilising them.

Anal Biochem. 2010 Feb 15;397(2):227-32. doi:10.1016/j.ab.2009.10.022

Pinto FL, Lindblad P.

A guide for in-house design of template-switch-based 5' rapid amplification of cDNA ends systems.

Uses desoxyriboguanosine tails for template switching (but the last one is blocked by a propyl group (C3 spacer).

Only RNAseH-minus reverse transcriptases will add more cap-templated nucleotides to the first strand cDNA in presence of manganese.