Sunflower: epigenetic drug treatment
One microgram of the extracted RNA was DNase digested for 60 min at 37C in a final volume of 10 l containing 0.5 ul of DNase I and 1 ul RDD Buffer (Qiagen, Germantown, United States) and incubated for 10 min at 75C with the addition of 1 ul EDTA of 25 M EDTA.2 g of DNase-treated total RNA was used for reverse transcription. In MmLV-catalyzed reactions RNA was primed poly-A specific hexamers from MINT cDNA kit (Evrogen, Moscow, Russia), according to the manufacturers instructions. During synthesis the optimal number of PCR cycles (20 cycles) was adapted to reach the exponential phase of amplification. For Nanopore sequencing, a library was prepared from ds-cDNA using the nanopore native barcoding genomic DNA SQK-NBD110-24 (Oxford Nanopore Technologies, Oxford, UK) with some modification in the process of using NEBNext Companion Module for Oxford Nanopore Technologies Ligation Sequencing (New England Biolabs, Massachusetts, USA). ~ 500 ng of each ds-cDNA in 24 l was mixed with 1.75 l NEBNext FFPE DNA Repair Buffer, 1.75 l Ultra II End-prep reaction buffer, 1.5 l Ultra II End-prep enzyme mix, 1 l NEBNext FFPE DNA Repair Mix and incubated at the thermal cycler for 5 min at 20C and for 5 min at 65C. The DNA samples were transferred to new tube and added extra 30 l H2O to reach the required volume for the next preparations of the DNA library. Each end-prepped sample was barcoded 2.5 l native barcodes from SQK-NBD110-24 kit (Oxford Nanopore Technologies, Oxford, UK). After purification 4 barcoded DNAs were combined to a final volume of 65 l with further ligation of adapters for sequencing using Quick T4 DNA ligase (New England Biolabs, Massachusetts, USA) according to SQK-LSK109 kit (Oxford Nanopore Technologies, Oxford, UK).