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)
Data files
Oct 20, 2023 version files 44.75 GB
Abstract
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).
README: 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
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.
Methods
RNA-seq:
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.
ChIP-seq:
SAB23 was fused with GFP and transformed into DJ via Agrobacterium tumefaciens (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.