Total RNAs were isolated and separated on 10% polyacrylamide–urea gel, transferred to nylon membrane (GeneScreen Plus; PerkinElmer) using IBlot (Invitrogen). The membrane was then UV cross-linked and vacuum dried at 80°C for 2 h. It was hybridized with specific 32P-labeled DNA oligos as described previously (Lamichhane et al. 2011 (link)). Blots were exposed to a phosphorimager screen, scanned, and quantified using a Phosphorimager FLA-3000 (Fujifilm). The PHA6 assay was used to assess the level of i6A37 modification (Lamichhane et al. 2013a (link),b (link); Yarham et al. 2014 (link)). Quantification of the efficiency of i6A37 modification used the formula: % modification = [1 − (ACLtit1+/TSLtit1+)/(ACLtit1-Δ/TSLtit1-Δ)] × 100, where “ACL” indicates the ACL probe and “TSL” indicates the TSL probe. As previously described, this formula includes an internal normalization from the tit1-Δ samples (Lamichhane et al. 2013a (link),b (link); Yarham et al. 2014 (link)). However, it should be noted that given the principle of PHA6 assay, which depends on the amount of hypomodified adenosine (A37), the modification ratio calculated by this formula thus represents the relative amount of i6A, but not absolute modification rate.