Nucleotrap mrna mini kit

MeRIP-seq was performed largely as described by Dominissini et al. ^{36 (link)}. Total RNA from HEL cells was isolated by Trizol (ThermoFisher) and the Direct-zol RNA kit (Zymo Research). Poly(A) RNA was then isolated with the NucleoTrap mRNA Mini kit (Macherey–Nagel) yielding ~5 µg of polyA RNA per replicate. The polyA RNA was then fragmented using the NEBNext Magnesium RNA Fragmentation Module (NEB) for 7 min at 94 °C yielding RNA fragments of ~125 bp. Fragmented RNA was incubated with 5 µg m⁶A antibody (Millipore Sigma, ABE572) for 2 h, followed by immunoprecipitation and phenol:chloroform cleanup and ethanol precipitation. Sequencing libraries were generated using the KAPA Biosystems Stranded RNA-Seq Kit (Roche), were quantified using a Qubit Fluorometer (ThermoFisher), and the size distribution was checked using TapeStation 4200 (Agilent Technologies). 50 bp paired-end reads were sequenced on an Illumina HiSeq 2000.

Total RNA was isolated from mock inoculated and infected leaf tissues by using the Trizol reagent (Invitrogen, Carlsbad, CA) treated with DNase-I (Sigma-Aldrich, USA) to eliminate traces of genomic DNA and purified using the RNeasy Plant Mini Kit (Qiagen, USA) following manufacturer’s instruction. Integrity of the isolated RNA samples were assessed by agarose gel electrophoresis and purity and quantity of individual samples were determined spectrophotometrically (NanoDrop 1000 Spectrophotometer, Thermo Scientific, USA). Subsequently, an equal amount of total RNA (50 μg from each time point) were pooled for each sample and used as a starting material for mRNA extraction. mRNA was purified from the pooled total RNA samples using the NucleoTrap mRNA Mini kit (Macherey-Nagel, Germany).

PolyA+ RNA was extracted at the fourth day time-point from U2OS cells transfected with a MfPV minicircle genomes. The RNA samples were prepared by isolation of total RNA by TriReagent (Molecular Research Center, Inc.) followed by polyA+ RNA purification using NucleoTrap mRNA Mini kit (MACHEREY-NAGEL GmbH) according to the manufacturer’ instructions. 1 μg of polyA+ RNA was used as a template for 5' or 3' RACE, respectively, performed with the SMARTer RACE 5’/3’ Kit (Takara Bio USA, Inc.) according to the manufacturer's instructions. For RT-PCR, 1 μg cDNA first-strand synthesis was conducted using the SuperScript IV First-Strand Synthesis System (Thermo Fischer Scientific, USA). The Phusion Hot Start II DNA polymerase (Thermo Fischer Scientific, USA) was used to amplify the RACE and RT-PCR products that were purified from agarose gel, cloned and fully sequenced as single clones (data in S1 and S2 Tables). The positions of the MfPV genome specific primers (GSPs) used for the RACE and RT-PCR amplifications are listed in Table 1.