SHAPE selection (SHAPES) enrich for RNA structure signal in SHAPE sequencing-based probing data
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SHAPE selection (SHAPES) enrich for RNA structure signal in SHAPE sequencing-based probing data. / Poulsen, Line Dahl; Kielpinski, Lukasz Jan; Salama, Sofie R; Krogh, Anders; Vinther, Jeppe.
In: R N A, Vol. 21, No. 5, 2015, p. 1042-1052.Research output: Contribution to journal › Journal article › Research › peer-review
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T1 - SHAPE selection (SHAPES) enrich for RNA structure signal in SHAPE sequencing-based probing data
AU - Poulsen, Line Dahl
AU - Kielpinski, Lukasz Jan
AU - Salama, Sofie R
AU - Krogh, Anders
AU - Vinther, Jeppe
N1 - © 2015 Poulsen et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.
PY - 2015
Y1 - 2015
N2 - Selective 2' Hydroxyl Acylation analyzed by Primer Extension (SHAPE) is an accurate method for probing of RNA secondary structure. In existing SHAPE methods, the SHAPE probing signal is normalized to a no-reagent control to correct for the background caused by premature termination of the reverse transcriptase. Here, we introduce a SHAPE Selection (SHAPES) reagent, N-propanone isatoic anhydride (NPIA), which retains the ability of SHAPE reagents to accurately probe RNA structure, but also allows covalent coupling between the SHAPES reagent and a biotin molecule. We demonstrate that SHAPES-based selection of cDNA-RNA hybrids on streptavidin beads effectively removes the large majority of background signal present in SHAPE probing data and that sequencing-based SHAPES data contain the same amount of RNA structure data as regular sequencing-based SHAPE data obtained through normalization to a no-reagent control. Moreover, the selection efficiently enriches for probed RNAs, suggesting that the SHAPES strategy will be useful for applications with high-background and low-probing signal such as in vivo RNA structure probing.
AB - Selective 2' Hydroxyl Acylation analyzed by Primer Extension (SHAPE) is an accurate method for probing of RNA secondary structure. In existing SHAPE methods, the SHAPE probing signal is normalized to a no-reagent control to correct for the background caused by premature termination of the reverse transcriptase. Here, we introduce a SHAPE Selection (SHAPES) reagent, N-propanone isatoic anhydride (NPIA), which retains the ability of SHAPE reagents to accurately probe RNA structure, but also allows covalent coupling between the SHAPES reagent and a biotin molecule. We demonstrate that SHAPES-based selection of cDNA-RNA hybrids on streptavidin beads effectively removes the large majority of background signal present in SHAPE probing data and that sequencing-based SHAPES data contain the same amount of RNA structure data as regular sequencing-based SHAPE data obtained through normalization to a no-reagent control. Moreover, the selection efficiently enriches for probed RNAs, suggesting that the SHAPES strategy will be useful for applications with high-background and low-probing signal such as in vivo RNA structure probing.
U2 - 10.1261/rna.047068.114
DO - 10.1261/rna.047068.114
M3 - Journal article
C2 - 25805860
VL - 21
SP - 1042
EP - 1052
JO - RNA
JF - RNA
SN - 1355-8382
IS - 5
ER -
ID: 136791443