Sequel sequencing; Raw reads: DE01087_REF_CLR_SMRTcell23
Prior to the HMW extraction the reference plant was kept for 48 hours in the dark to reduce the starch accumulation. We harvested ca. 30 g of young leaf material and ground it in liquid nitrogen. Nuclei isolation was performed according to the protocol of Workman et al. (2018) with the following modifications: we used 16 reactions, each with 1 g input material in a 20 ml nuclear isolation buffer. The filtered cellular homogenate was centrifuged at 3500 x g, followed by 3x washes in nuclear isolation buffer. The isolated plant cell nuclei were resuspended in 60 μl Proteinase K (Cat. No. 19131, Qiagen). For HMW-DNA recovery, the Nanobind Plant Nuclei Big DNA Kit (SKU NB-900-801-01, Circulomics) was used. In total, we got approximately 80 μg of HMW-DNA, which was subjected to needle shearing once (FINE-JECT 26Gx1'' 0.45x25mm, LOT 14-13651). A 75-kb template library was prepared with the SMRTbell Express Template Preparation Kit 2.0, and size-selected with the BluePippin system (SageScience) with 15-kb cutoff and a 0.75% agarose, 1-50kb cassette (BLF7510, Biozym) according to the manufacturer's instructions (P/N 101-693-800-01, Pacific Biosciences, California, USA). The library was sequenced on a Sequel I system (Pacific Biosciences) using the Binding Kit 3.0. and MagBead loading. In total, we sequenced 18 SMRT cells of 10 hours and 8 SMRT cells of 20 hours movie time.