DOI: 10.5061/dryad.k0p2ngfd3
title: RNA seq of 4-week-old SAB23KO mutants and wild-type plants soaked for 0 or 1 day, and 6 ChIP libraries sequence (two DJ and four SAB23-GFP)

description: This dataset contains raw data related to the article published in the
 Journal of Experimental Botany, including RNAseq of 4-week-old SAB23KO
 mutants and wild-type plants soaked for 0 or 1 day, and 6 ChIP libraries
 sequences (two DJ and four SAB23-GFP).
description: <strong>RNA-seq:</strong>
 Four-week-old SAB23KO mutant and WT plants were subjected to
 submergence for 0  or 1 d, and fully expanded leaves were collected
 individually for RNA-seq. Approximately 0.5 g of leaves from six seedlings
 of each genotype were sampled; all experiments were performed with three
 biological replications. The leaf samples were ground to a fine powder in
 liquid nitrogen, and total RNA was isolated using the TRIzol reagent
 (Invitrogen, Carlsbad, CA, USA). In total, 12 RNA samples were prepared
 and submitted to the Beijing Genomics Institute (BGI) for
 sequencing.
 <strong>ChIP-seq:</strong> SAB23
 was fused with GFP and transformed into DJ via <em>Agrobacterium
 tumefaciens</em> (EHA105). The transgenic lines were generated by
 Wuhan Biorun Biotechnology Co. Three biological replications were
 performed for each sample. Approximately 5 g of leaves were collected from
 four-week-old SAB23-GFP and DJ seedlings (as a negative control).
 Expression of SAB23-GFP was verified by protein gel blotting using
 anti-GFP antibody (Abcam, AB290) at a dilution of 1:1000 (v/v) in
 Tris-buffered saline containing 5% nonfat milk powder. The leaves were
 immediately cross-linked for 15 min in buffer containing 1% (v/v)
 formaldehyde under vacuum and quenched by addition of glycine to a final
 concentration of 0.1 M and infiltration for 5 min. After three washes with
 distilled water (4°C), the cross-linked tissues were dried with paper
 towels and frozen in liquid nitrogen. Frozen tissues were ground
 thoroughly to a fine powder, which was transferred to a precooled 50-ml
 tube with 20 ml of cold complete extraction buffer 1 (10 mM Tris-HCl [pH
 8.0], 0.4 M sucrose, 10 mM MgCl, 1 mM phenylmethylsulfonyl fluoride
 [PMSF], 5 mM β-mercaptoethanol, and Plant Protease Inhibitor Cocktail
 [P9599, Sigma–Aldrich]). Homogenized tissues were centrifuged for 20 min
 at 1000 g and 4°C. The pellets were washed five times with 5 ml of
 complete extraction buffer 2 (10 mM Tris-HCl [pH 8.0], 0.25 M sucrose, 10
 mM MgCl2, 1% [v/v] Triton X-100, 1 mM PMSF, 5 mM β-mercaptoethanol, and
 Plant Protease Inhibitor Cocktail) and once with extraction buffer 3 (10
 mM Tris-HCl [pH 8.0], 1.7 M sucrose, 2 mM MgCl2, 0.15% [v/v] Triton X-100,
 1 mM PMSF, 5 mM β-mercaptoethanol, and Plant Protease Inhibitor Cocktail).
 The washed pellets were resuspended in 300 µl of nuclei buffer (50 mM
 Tris-HCl [pH 8.0], 10 mM EDTA, 1% SDS, 1 mM PMSF, and Plant Protease
 Inhibitor Cocktail), and the suspension was treated with a Bioruptor
 (Diagenode, Belgium) for 8 to 10 cycles with settings of 30 s ON/30 s OFF
 at 4°C until the average genomic DNA fragment size was ~200–500 bp. The
 sonicated sample was centrifuged for 10 min at 12,000 g and 4°C, and the
 supernatant was collected and used for chromatin isolation. Fragmented
 chromatin (150 μl) was diluted with 1350 μl of ChIP dilution buffer (50 mM
 Tris-HCl [pH 8.0], 167 mM NaCl, 1.1% Triton X-100, 0.11% sodium
 deoxycholate, and Plant Protease Inhibitor Cocktail). Dynabeads Protein A
 magnetic beads (Invitrogen) were blocked in blocking buffer (200 μg
 glycogen, 200 μg BSA, and 200 μg yeast tRNA in 1 ml ChIP dilution buffer).
 Isolated chromatin was precleared by incubating with 40 μl of blocked
 Dynabeads for 2 h at 4°C. Approximately 2 μg of anti-GFP antibody
 (Invitrogen, A11122) was used in the immunoprecipitation reactions at 4°C
 overnight. The bound chromatin complex was captured by 40 μl blocked
 Dynabeads and washed successively with low-salt wash buffer (50 mM
 Tris-HCl [pH 8.0], 150 mM NaCl, 1 mM EDTA, 0.1% SDS, 0.1% sodium
 deoxycholate, 1% Triton X-100, and 1× Complete Protease Inhibitor
 Cocktail), high-salt wash buffer (50 mM Tris-HCl [pH 8.0], 500 mM NaCl, 1
 mM EDTA, 0.1% SDS, 0.1% sodium deoxycholate, 1% Triton X-100, and 1×
 Complete Protease Inhibitor Cocktail), lithium chloride buffer (250 mM
 LiCl, 1% Nonidet P-40, 1% sodium deoxycholate, 1 mM EDTA, and 10 mM
 Tris-HCl [pH 8.0]), and Tris-EDTA buffer (twice). The bead pellets were
 incubated at 65°C overnight in 200 μl of elution buffer (10 mM Tris-HCl
 [pH 8.0], 300 mM NaCl, 5 mM EDTA, 0.5% SDS, 20 mg Proteinase K, and 3 μl
 RNaseA). ChIP DNA was isolated by phenol–chloroform extraction and
 isopropanol precipitation with 40 μg of glycogen (Roche) and 240 mM sodium
 acetate. The Ovation Low Input DR Kit (NuGEN Technologies, San Carlos, CA,
 USA) was used for ChIP-seq library construction. A
 total of six ChIP libraries (two DJ and four SAB23) were sequenced on an
 Illumina HiSeq 2000 sequencer (Illumina Inc., USA), generating 150 bp
 paired-end reads.
description: # RNA seq of 4-week-old SAB23KO mutants and wild-type plants soaked for 0
 or 1 day，and 6 ChIP libraries sequence (two DJ and four SAB23-GFP)
 [https://doi.org/10.5061/dryad.k0p2ngfd3](https://doi.org/10.5061/dryad.k0p2ngfd3) This dataset contains raw data related to the submitted article, includingRNA seq  of 4-week-old SAB23KO mutants and wild-type plants soaked for 0 or 1 day，and 6 ChIP libraries sequence (two DJ and four SAB23-GFP). 6 ChIP libraries sequence (two DJ and four SAB23): SAB23 was fused with GFP and transformed into DJ via Agrobacterium tumefaciens (EHA105). The transgenic lines were generated by Wuhan Biorun Biotechnology Co. ChIP was carried out according to Dong et al. (2019), and three biological replications were performed for each sample. Approximately 5 g of leaves were collected from four-week-old SAB23-GFP and DJ seedlings (as a negative control). Expression of SAB23-GFP was verified by protein gel blotting using an anti-GFP antibody (Abcam, AB290) at a dilution of 1:1000 (v/v) in Trisbuffered saline containing 5% nonfat milk powder. The leaves were immediately cross-linked for 15 min in buffer containing 1% (v/v) formaldehyde under vacuum and quenched by addition of glycine to a final concentration of 0.1 M and infiltration for 5 min. After three washes with distilled water (4°C), the cross-linked tissues were dried with paper towels and frozen in liquid nitrogen. Frozen tissues were ground thoroughly to a fine powder, which was transferred to a precooled 50 ml tube with 20 ml of cold complete extraction buffer 1 (10 mM Tris-HCl [pH 8.0], 0.4 M sucrose, 10 mM MgCl, 1 mM phenylmethylsulfonyl fluoride [PMSF], 5 mM β-mercaptoethanol, and Plant Protease Inhibitor Cocktail [P9599, Sigma-Aldrich]). Homogenized tissues were centrifuged for 20 min at 1000 g and 4°C. The pellets were washed five times with 5 ml of complete extraction buffer 2 (10 mM Tris-HCl [pH 8.0], 0.25 M sucrose, 10 mM MgCl2, 1% [v/v] Triton X-100, 1 mM PMSF, 5 mM β-mercaptoethanol, and Plant Protease Inhibitor Cocktail) and once with extraction buffer 3 (10 mM Tris-HCl [pH 8.0], 1.7 M sucrose, 2 mM MgCl2, 0.15% [v/v] Triton X-100, 1 mM PMSF, 5 mM β-mercaptoethanol, and Plant Protease Inhibitor Cocktail). The washed pellets were resuspended in 300 µl of nuclei buffer (50 mM Tris-HCl [pH 8.0], 10 mM EDTA, 1% SDS, 1 mM PMSF, and Plant Protease Inhibitor Cocktail), and the suspension was treated with a Bioruptor (Diagenode, Belgium) for 8 to 10 cycles with settings of 30 s ON/30 s OFF at 4°C until the average genomic DNA fragment size was ~200-500 bp. The sonicated sample was centrifuged for 10 min at 12,000 g and 4°C, and the supernatant was collected and used for chromatin isolation. Fragmented chromatin (150 μl) was diluted with 1350 μl of ChIP dilution buffer (50 mM Tris-HCl [pH 8.0], 167 mM NaCl, 1.1% Triton X-100, 0.11% sodium deoxycholate, and Plant Protease Inhibitor Cocktail). Dynabeads Protein A magnetic beads (Invitrogen) were blocked in blocking buffer (200 μg glycogen, 200 μg BSA, and 200 μg yeast tRNA in 1 ml ChIP dilution buffer). Isolated chromatin was precleared by incubating with 40 μl of blocked Dynabeads for 2 h at 4°C. Approximately 2 μg of anti-GFP antibody (Invitrogen, A11122) was used in the immunoprecipitation reactions at 4°C overnight. The bound chromatin complex was captured by 40 μl blocked Dynabeads and washed successively with low-salt wash buffer (50 mM Tris-HCl [pH 8.0], 150 mM NaCl, 1 mM EDTA, 0.1% SDS, 0.1% sodium deoxycholate, 1% Triton X-100, and 1×Complete Protease Inhibitor Cocktail), high-salt wash buffer (50 mM Tris-HCl [pH 8.0], 500 mM NaCl, 1 mM EDTA, 0.1% SDS, 0.1% sodium deoxycholate, 1% Triton X-100, and 1×Complete Protease Inhibitor Cocktail), lithium chloride buffer (250 mM LiCl, 1% Nonidet P-40, 1% sodium deoxycholate, 1 mM EDTA, and 10 mM Tris-HCl [pH 8.0]), and Tris-EDTA buffer (twice). The bead pellets were incubated at 65°C overnight in 200 μl of elution buffer (10 mM Tris-HCl [pH 8.0], 300 mM NaCl, 5 mM EDTA, 0.5% SDS, 20 mg Proteinase K, and 3 μl RNaseA). ChIP DNA was isolated by phenol-chloroform extraction and isopropanol precipitation with 40 μg of glycogen (Roche) and 240 mM sodium acetate. The Ovation Low Input DR Kit (NuGEN Technologies, San Carlos, CA, USA) was used for ChIP-seq library construction. The RNA-seq of Four-week-old SAB23KO mutant and WT plants which were subjected to submergence for 0 or 1 d: Four-week-old SAB23KO mutant and WT plants were subjected to submergence for 0 or 1 d, and fully expanded leaves were collected individually for RNA-seq. Approximately 0.5 g of leaves from six seedlings of each genotype were sampled; all experiments were performed with three biological replications. The leaf samples were ground to a finpowder in liquid nitrogen, and total RNA was isolated using the TRIzol reagent (Invitrogen, Carlsbad, CA, USA). In total, 12 RNA samples were prepared and submitted to the Beijin Genomics Institute (BGI) for sequencing.