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Sample GSM7821646

Query DataSets for GSM7821646

Status

Public on Oct 04, 2023

Title

ChIP-Seq Sorghum bicolor: Btx623 whole root replicate 1 under high phosphorous condition and K4me3 antibody

Sample type

SRA

Source name

whole root

Organism

Sorghum bicolor

Characteristics

tissue: whole root
treatment: normal phosphorus

Extracted molecule

genomic DNA

Extraction protocol

RNA-seq: Root tissue samples were ground in liquid nitrogen and then the RNA was isolated in Trizol reagent (ThermoFisher) followed by purifica- tion using Zymo-spin columns (Directzol RNA kit; Zymo Research). Column-bound RNA was treated with DNase 1 and finally eluted with 50 μl of DNase/RNase-free water. RNA quality was determined using the Bioanalyzer hardware with the Agilent RNA 6000 Nano Kit (Agilent). Poly-A RNA was isolated from total RNA using Dynabeads (ThermoFisher). Libraries for sequencing were created using the ScriptSeq RNA-seq prep kit (Illumina) following manufacturer protocols. Final libraries were amplified with 17 cycles of PCR and assessed on the Bioanalyzer with the High Sensitivity DNA kit (Agilent). Bisulphite-seq: Bisulfite-sequencing was done as described in (Li et al., 2014). The root apices (first two cm) of all primary and lateral roots were excised from plants grown as described above after 10 days of growth on SP and LP media. The root samples from 10 plants constituted one bio- logical replicate. DNA was isolated from root sections and sheared into fragments between 200 and 300 bp in length. These fragments underwent end repair, dA tailing, and ligation to methylated adaptors for subsequent bisulfite conversion. Libraries were PCR amplified, purified, and qual- ity controlled via Agilent DNA 1000 chip. Sequencing was done on a HiSeq 2500 v4 with 125 bp paired-end reads. ChIP-seq: The entire root systems were harvested for ChIP-seq library prepara- tion. The root tissue was cut into sections and fixed in 10-mM dimethyl adipimate (DMA; Sigma-Aldrich) while applying a vacuum. Samples were ground in liquid nitrogen and then placed in extraction buffer, filtered through miracloth and then a 30-mm CellTrics filter (CellTrics). Nuclei were isolated and then chromatin was sheared in 130-ml tubes (Covaris). Shearing was performed on a Covaris S220 sonicator for 5 min at a cycle/burst of 200, peak power of 175, and duty factor of 10. Immunoprecipitation was performed with anti- bodies for H3K27me3 (Millipore), H3K4me3 (Abcam), H3 (Abacam) for input control, and Protein A Dynabeads (ThermoFisher) for input control. Crosslinking was reversed and samples were purified using Chip DNA Clean and Concentrator Kit (Zymo Research).
RNA-seq: Libraries for sequencing were created using the ScriptSeq RNA-seq prep kit (Illumina) following manufacturer protocols. Final libraries were amplified with 17 cycles of PCR and assessed on the Bioanalyzer with the High Sensitivity DNA kit (Agilent). All root tis- sue libraries that were sequenced comprised two biological replicates. Bisulphite-seq: DNA was isolated from root sections and sheared into fragments between 200 and 300 bp in length. These fragments underwent end repair, dA tailing, and ligation to methylated adaptors for subsequent bisulfite conversion. Libraries were PCR amplified, purified, and qual- ity controlled via Agilent DNA 1000 chip. Chip-seq: Libraries were created using the Nextflex ChIP-Seq library prep kit (Bioo Scien- tific) with the NextFlex ChIP-seq Barcodes. Sequencing was done on a HiSeq 2500 v4 with 125 bp paired-end reads

Library strategy

ChIP-Seq

Library source

genomic

Library selection

ChIP

Instrument model

Illumina HiSeq 2000

Description

G3_P_K4me3_rep1_tagDir_inp_controlled.HisTight.bw
G3_P_K4me3_rep2_tagDir_inp_controlled.HisTight.bw

Data processing

RNA-seq: Paired-end fastq files were trimmed for quality with Trimmomatic and then merged with Samtools before aligning to the v3.4.1 S. bicolor genome with Kallisto package in rStudio v3.6.1. Differential gene expression was deter- mined through DESeq2 after importing Kallisto abundance files with tximport package. Dimensional analysis was performed using the factoextra package. Heatmaps and hierarchical clustering were per- formed with the heatmpap.2 package in rStudio.
ChIP-seq: Paired-end fastq files were trimmed for quality with Trimmomatic and then mapped to the v3.4.1 S. bicolor genome with BWA (bwa mem -q 30, -f 2 -ubhS). Sorted bam files were marked for duplicates with Picard and then merged with Samtools. Tag directories creation and peak calling, peak enrichment comparison was performed through the Homer package. Overlapped peaks were identified through Bedtools (intersectBed). Peak annotation was performed using the annotatePeaks module of the Homer software. Samtools was used to obtain flagstat metrics and perform various file conversion and handling. Various visualization and file creation was performed using the deepTools package (Ramírez et al., 2016); aligned bam files (that were input-controlled) were converted to bigWig format for generating computeMatrix files for plotHeatmap and plotEnrichment outputs. k-means cluster number was determined by selecting the result that generated the distinct read coverage within and around gene models while minimizing clus- ter overlap.
Bisulphite-Seq: Reads were trimmed and quality controlled using Trim Galore and FASTQC respectively (http://www.bioinformatics.babraham.ac.uk/projects/index.html). Reads were aligned and extracted with Bismark (Krueger & Andrews, 2011). Differential methylation quantification and visualiza- tions were done with MethylKit (Akalin et al., 2012). Additional visual- izations were performed with deepTools (Ramírez et al., 2016) after converting Bismark-aligned bam files to bigWig and then generating computeMatrix files for plotHeatmap and plotEnrichment outputs.
Assembly: Sorghum Bicolor BTx623 v 3.1
Supplementary files format and content: RNA-seq: abundance files (*_abundance.tsv) from a kallisto quant run of trimmomatic-trimmed fastq files. These files are used for input into a differential expression analysis program (DESeq2 or edgeR).
Supplementary files format and content: ChIP-seq: these are bigwig files after processing with the Homer package. Aligned read files were marked for duplicates with Picard and then merged with Samtools. Subsequently, tag directories, peak calling, and peak enrichment were performed with Homer. These files are those outputs using the 'genomic DNA as control' samples as input controls for the calling.
Supplementary files format and content: Bisulphite-Seq: the *_all_differential_methylated_regions.csv files were generated from the MethylKit package after aligning the fastq files with Bismark. These files contain differential methylated sites for CPG, CHG, and CHH methylation using normal phosphorus growth conditions as the control. Statistical values for each call are included in the .csv files as well as a MethylKit score.

Submission date

Oct 04, 2023

Last update date

Oct 04, 2023

Contact name

Nicholas Gladman

Organization name

USDA

Department

ARS

Street address

1 Bungtown Rd