Novaseq 6000 sequencing platform

One hundred flies were collected on the third or fourth day with three biological replicates after being fed with or without erythritol for RNA extraction. Total RNA was extracted with the Trizol reagent and quantified using an Agilent 2100 bioanalyzer. RNA sequencing libraries were constructed using the NEBNext^® Ultra™ RNA Library Prep Kit for Illumina^® (New England Biolabs, Ipswich, MA, USA). mRNAs were enriched using Oligo(dT) magnetic beads, followed by random fragmentation in NEB Fragmentation Buffer. The first strand of cDNA was synthesized in the M-MuLV reverse transcriptase system using fragmented mRNA as templates and random oligonucleotides as primers. After RNA removal with RnaseH, the second strand of cDNA was synthesized using DNA polymerase I. Purified dscDNA was end-repaired, an adapter was added, the PCR was amplified, and then sequencing was carried out on the Illumina NovaSeq 6000 sequencing platform (Novogene Bioinformatics Technology Co., Ltd., Beijing, China). The data can be downloaded on GEO (accession number: GSE221267).

We harvested root samples of seedlings grown in paper rolls for 6 h, 24 h and 48 h after water deficit stress induction. We then separated the roots into three distinct root zones: root cap and meristem, elongation zone and differentiation zone and immediately froze them in liquid nitrogen. The boundaries between the meristematic zone and the elongation zone were estimated based on previously analyzed longitudinal sections by Kirschner et al. [31 (link)], where the transition started around 1 mm from the root tip, while the boundaries towards the differentiation zone were marked by the first appearance of root hairs. In total, we obtained 54 samples with three biological replicates for all treatment-by-root zone-by -time point combinations with a pool of 30 roots for each biological replicate. We extracted total RNA with the RNeasy Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions and assessed RNA quality and integrity with a NanoDrop (Thermo Fisher Scientific, Waltham, MA, USA) and a BioAnalyzer (Agilent RNA 6000 Nano Chip, Agilent Technologies, Santa Clara, CA, USA). All samples exceeded a RNA integrity number value of 8.1. The RNA samples were sequenced on a NovaSeq 6000 sequencing platform (Novogene, Cambridge, UK) using a paired-end 150 bp strategy.