Time-series RNA-Seq data of leaves with altered leaf longevity in the self-shading and reproductive-sink-removal experiments of Arabidopsis halleri sbusp. gemmifera.. Arabidopsis halleri subsp. gemmifera

In a natural population of Arabidopsis halleri subsp. gemmifera, we conducted two sets of experiments that evaluated responses of leaf longevity. The RNA-Seq data from the four sets of experiments are deposited here. In the first two sets of experiments, shaded and exposed treatments were applied to leaves that emerged during growth seasons and over-wintering (the self-shading experiments for the GS and OW cohort). In the other two sets of experiments, sink-removal (Sink_minus) treatment and intact control (Sink_plus) were applied to leaves that emerged during growth seasons and overwintering seasons (the sink-removal experiments for the GS and OW cohort). Designs of RNA sampling differed between the self-shading and sink-removal experiments, because two treatments were applied to different leaves within a plant and to different plants within a plant pair (explained above in Sink-removal experiment) in the former and the latter experiments, respectively.In the GS self-shading experiment, six and five treatment pairs were sampled for each of five sampling times which were 0, 2, 4, 6, and 8 weeks after treatment initiation (23 July, 6 August, 20 August, 3 September, and 17 September, respectively). Four pairs of samples per time point were further used for RNA-seq. In the OW self-shading experiment, five treatment pairs were sampled for each of four sampling times which were 0, 4, 8, and 12 weeks after treatment initiation (26 January, 23 February, 23 March, and 20 April, respectively). We analysed five pairs of samples per time point for RNA-seq. In the GS sink-removal experiments, samples were collected at seven time points at 1 week before the treatment, and 0, 1, 2, 4, 6, and 8 weeks after the treatment initiation (28 July, 4, 11, and 18 August, and 1, 15, and 29 September, respectively). Either two (at 2, 6, and 8 weeks after the treatment initiation) or three (at the other four time points) treatment pairs were sampled at each sampling. All samples obtained were used for RNA-seq. In the OW sink-removal experiment, samples were collected at eight time points at 5 weeks before treatment, and 0, 1, 2, 4, 6, 8, and 12 weeks after the start of treatment (15 February, 22 and 29 March, 5 and 19 April, 2 and 17 May, and 14 June, respectively). Either three (0 and 6 weeks after the start of the treatment) or four (other time points) replicates per treatment were sampled at each sampling. Pairwise samples were use up to 4 weeks after the treatment, and only the sink_minus treatment was sampled at later time points. In the sink_plus treatment, all leaves had withered by 6 weeks after the start of the treatment, and therefore leaves were not sampled after this point. All samples collected were used for RNA-seq. For plants all tagged leaves were withered before RNA sampling, we did not sample leaves. All samplings were performed at noon and sampled leaves were immediately preserved in 1.5 ml microtubes with 500 µl RNAlater (Thermo Fisher Scientific, Massachusetts, U.S.A., #AM7021r) on ice and returned to the laboratory. After keeping them overnight in a refrigerator at 4 degree, they were stored at -20 degree in a freezer until they were used for further analyses. Leaf samples were homogenised in lysis/binding buffer using a multi-beads shocker (Yasui Kikai, Osaka, Japan). The mRNA was isolated directly from the homogenate using streptavidin magnetic beads (New England Biolabs, Ipswich, MA, USA, #S1420S) and 5ʹ biotinylated polyT oligonucleotide. RNA libraries were prepared using the Breath Adapter Directional sequencing (BrAD-seq) method for strand-specific 3ʹ digital gene expression quantification. Briefly, the mRNA was heat-fragmented and primed with a 3ʹ adapter-containing oligonucleotide primer targeting the polyA tail of the mRNA. The cDNA was synthesised using RevertAid Reverse Transcriptase (Thermo Fisher Scientific, #EP0441) using a Veriti Dx Thermal Cycler (Thermo Fisher Scientific). The 5ʹ adapter was added by strand-specific breath capture and the second strand was synthesised using DNA Polymerase I (Thermo Fisher Scientific, #EP0041). Final PCR enrichment was performed using oligonucleotides containing the full adapter sequence with a unique index for each sample. The PCR products were purified and size selected using AMpure XP beads (Beckman Coulter, Brea, CA, USA, #A63881). The size distribution and concentration of the library were measured using a Model 2100 Bioanalyzer (Agilent Technology, Palo Alto, CA, USA) and QuantiFluor DNA System (Promega, Madison, WI, USA) with an Infinite 200 PRO microplate photometer (TECAN, Basel, Switzerland), respectively. Products from 40, 40, 36 and 48 samples from GS self-shading and OW self-shading and GS sink-removal and OW sink-removal respectively were pooled as two sets of libraries for Illumina sequencing systems. The four libraries were sequenced using two lanes on a HiSeq 2500 instrument (Illumina). The original BrAD-Seq protocol was modified to use KAPA HiFi HotStart ReadyMix (Kapa Biosystems, Woburn, MA, USA, #KK2062) for the final PCR.