DOI: 10.5061/dryad.b2rbnzskc
title: RNA-seq and RT-qPCR data showing MDF role in RNA splicing and gene expression control in Arabidopsis

description: Plants respond to environmental stresses through controlled stem cell
 maintenance and meristem activity. One level of transcriptional control is
 RNA alternative splicing. However the mechanistic link between stress,
 meristem function and RNA splicing is poorly understood.
 The MERISTEM-DEFECTIVE (MDF)/DEFECTIVELY ORGANIZED
 TRIBUTARIES (DOT2) gene of Arabidopsis encodes a
 SR-related family protein, required for meristem function and leaf
 vascularization, and is the likely orthologue of the human SART1 and yeast
 snu66 splicing factors. MDF is required for the correct splicing and
 expression of key transcripts associated with root meristem function. We
 identified RSZ33 and ACC1, both known to
 regulate cell patterning, as splicing targets required for MDF function in
 the meristem. MDF expression is modulated by osmotic and
 cold stress, associated with differential splicing and specific isoform
 accumulation and shuttling between nucleus and cytosol, and acts in part
 via a splicing target SR34. We propose a model in which MDF
 controls splicing in the root meristem to promote stemness and repress
 stress response and cell differentiation pathways.
description: <strong>RNA extraction and
 sequencing</strong>
 RNA was extracted from three
 independent biological replicates using 7-day-old seedlings (ca. 100 mg
 tissue) grown on half-strength MS10 medium using the Sigma-Aldrich Plant
 Total RNA Kit (catalog number STRN50), with the On-Column DNase I
 Digestion Set (catalog
 Number: DNASE10-1SET) to
 eliminate any residual DNA molecules. Plant tissue was ground in liquid
 nitrogen before incubation in a lysis solution containing
 2-mercaptoethanol at 65°C for 3 minutes. The solid debris was removed by
 centrifuging and 14 000 x g and column filtration before RNA was captured
 onto a binding column using the supplied binding solution, which helps
 preventing polysaccharide and genomic DNA from clogging the column. Most
 DNA was removed by wash solutions, and any trace of residual DNA was
 removed by DNase on the column. Then purified RNA was eluted using
 RNAase-free water. RNA sequencing from three biological replicate
 samples was carried out on an Illumina HiSeq 2500 System with the library
 prepared using the Illumina TruSeq Stranded Total RNA with Ribo-Zero Plant
 Sample Preparation kit (catalog Number:
 RS-122-2401). Ribosomal
 RNA (rRNA) was removed from isolated total RNA using biotinylated,
 target-specific oligos on rRNA removal beads. Purified RNA was quality
 checked using a TapeStation 2200 (Agilent Technology) with High Sensitivity
 RNA ScreenTape (catalog Number: 5067-5579), and the mRNA was fragmented into 120-200 bp
 sequences with a median size of 150 bp. Fragmented mRNA was used as a
 template to synthesise first-strand cDNA using reverse transcriptase and
 random primers, followed by second-strand cDNA synthesis with DNA
 Polymerase I and RNase H. Newly synthesised cDNA had a single adenine base
 added with ligation of adaptors, before being purified and amplified by
 PCR to make the final library. Library quality control was performed again
 using a TapeStation with
 D1000 ScreenTape (catalog Number: 5067-5582).
 <strong>Pre-processing of RNA-seq data, differential
 expression and differential usage analysis.</strong>
 RNAseq data were processed and aligned against the TAIR10
 (EnsemblePlants) genome using TopHat and indexed with Samtools. DeSeq
 determined differential expression. Alternative splicing analysis was
 determined using RMats (p value of 0.05, a minimum of 10% inclusion
 difference). Alternative splicing events were visualised using Sashimi
 plots generated by the Integrative Genomics Viewer (IGV) (Robinson et al.,
 2011). <strong>Direct mRNA isolation and cDNA
 preparation for RT-qPCR or RT-PCR</strong>
 Seedlings were grown 7 days post-germination as described above.
 Roots and cotyledons were separated using a razor blade, and the material
 was frozen immediately in liquid nitrogen. Pools of seedlings were used to
 generate three separate biological samples. Each pool contained
 approximately 20 mg of root or cotyledon tissue. Total mRNA was extracted
 using Dynabeads®mRNA DIRECT™kit with Oligo(dT)25 labelled magnetic beads.
 Frozen tissue was ground with a sterile plastic micropestle and
 resuspended in 300 µl lysis buffer. The solution was then forced through a
 21-gauge needle in a 1ml syringe 3-5 times to shear any DNA and mixed with
 50 µl of Dynabeads Oligo(dT)25. The kit procedure was followed, with two
 final washes conducted. To ensure the complete removal of any genomic DNA
 in the subcellular fractionation experiments, this stage was followed by
 ezDnase™ treatment in a 10 µl volume (1µl ezDNASe™, 1 µl ezDNASe™ 10X
 buffer and 8 µl sterile H2O), 37˚C for 2 minutes followed by 1 µl DTT and
 5 minutes at 55˚C in a heat block. cDNA was prepared
 using a SuperScript®IV First-Strand synthesis system directly on the bead
 solution. For RT-PCR and RT-qPCR beads were washed in 20 µl 1 x SSIV
 buffer before resuspension in 12 µl sterile H2O with 1 µl dNTP 10 mM each
 mix and incubated for 5 minutes at 50 ˚C in a Proflex PCR machine (Applied
 Biosystems). Then the following were added 4 µl 10 x SSIV buffer, 1 µl
 ribonuclease inhibitor and 1 µl Superscript®IV reverse transcriptase were
 added. The mixture was mixed by pipetting and incubated for 10 minutes at
 50 ˚C, followed by 10 minutes at 80 ˚C, and then held at 4 ˚C. The 20 µl
 cDNA mix was stored at -20 and not eluted from the beads.
 Samples were checked for the presence of genomic DNA by PCR with
 Actin 2 primers ACT2 forward and reverse. A PCR reaction after 28 cycles
 with a Tm of 60 ˚C generated a 340 bp product if genomic DNA was a
 contaminant, 240 bp otherwise. All PCR and sequencing primers are listed
 in Table S3.
 <strong>RT-PCR</strong> 0.5 – 1 µl
 of cDNA/bead mix were used per PCR reaction. RT-PCR was performed
 with RSZ33 or ACC1 root-derived cDNA using Phusion™ (Thermofisher)
 high-fidelity polymerase. Relative levels of RSZ33 and ACC1 splice
 variants were determined using FIJI gel analysis software (Schindelin et
 al. 2012). Relative levels of cDNA per sample were determined using
 PCR-amplified PP2a transcript levels. 
description: Excel files are provided.