Illustra g50 columns

Okazaki fragments were purified and end-labeled essentially as described (25 (link)). Briefly, DNA concentration was normalized by Qubit. 650 ng of DNA per lane was treated with Klenow (exo-) (NEB) and α-³²P dCTP for 30 min at 37°C in NEBuffer 2. Unlabeled nucleotides were removed using Illustra G50 columns (GE Healthcare) and samples were separated for 5 h at 77 V in 1.3% alkaline agarose gels. DNA was transferred to a Nylon membrane overnight and visualized using a phosphorimager.
For in vitro ligation experiments, samples were incubated in 1× DNA ligase buffer (NEB). 2 μl of T4 DNA ligase (NEB) was added and samples were incubated for 90 min at room temperature before phenol extraction and end-labeling.
Okazaki fragment purification and sequencing was carried out as previously described (25 (link)). Paired-end sequencing (2 × 75 bp) was carried out on an Illumina Next-seq 500 platform.

RNA gel blot analysis was carried out as previously described (Quesada et al., 2003 (link)) with minor modifications. RPP7 mRNA was detected using a probe annealing to the second exon of the RPP7 (AT1G58602) gene (200 bp PCR product amplified with the following primers: Forward: 5′-TCGGGGACTACTACTACTCAAGA-3′ and Reverse: 5′-TCTTGATGGTGTGAAAGAATCTAGT-3′). β-TUBULIN mRNA was used as a loading control and visualised by a probe annealing to the third exon of the β-TUBULIN (AT1G20010) gene (550 bp PCR product amplified with the following primers: Forward: 5′- CTGACCTCAGGAAACTCGCG-3′ and Reverse: 5′- CATCAGCAGTAGCATCTTGG-3′). The probes were 5′ labelled using [γ-³²P]-ATP (Perkin Elmer) and DECAprime II DNA labelling kit (Thermo Fisher Scientific) and purified on illustra G-50 columns (GE Healthcare Life Sciences). mRNA isoforms were visualised and quantified using an Amersham Typhoon Gel and Blot Imaging System (GE Healthcare Bio-Sciences AB). The RiboRuler High Range RNA Ladder (Thermo Fisher Scientific), used to identify the approximate size of RNA bands, was first dephosphorylated using FastAP Thermosensitive Alkaline Phosphatase (Thermo Fisher Scientific) and then labelled with [γ-³²P]-ATP (Perkin Elmer) using T4 Polynucleotide Kinase (Thermo Fisher Scientific) before gel loading.