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Sample GSM5941875

Query DataSets for GSM5941875

Status

Public on Jul 26, 2022

Title

H2B8 ChIP-seq input rep 2 (50% PEG drought)

Sample type

SRA

Source name

p35S::H2B.8-eGFP seedling

Organism

Arabidopsis thaliana

Characteristics

ecotype: Col-0
genotype: p35S::H2B.8-eGFP
cell type: ten-day-old seedlings
chip antibody: none

Growth protocol

Plants were grown under long day (16 hr light; 8 hr dark) conditions at 22 °C.

Extracted molecule

genomic DNA

Extraction protocol

Ten-day-old seedlings of a p35S::H2B.8-eGFP line were ground with a pestle and mortar in liquid N2 and homogenized in nuclei isolation buffer (0.25 M sucrose, 15 mM PIPES pH 6.8, 5 mM MgCl2, 60 mM KCl, 15 mM NaCl, 1 mM CaCl2, 0.9% Triton X-100, 1 mM PMSF, 1x proteinase inhibitor Cocktail). Nuclei were separated from debris by filtering through two layers of miracloth. Chromatin was fragmented by micrococcal nuclease (MNase, New England Biolabs) digestion. For IP samples, fragmented chromatin was immunoprecipitated with GFP-Trap beads. For input, no immunoprecipitation was undertaken. Protein and RNA was removed by digestion with Proteinase K and RNase A, respectively. Phenol-chloroform extraction was used to obtain DNA.
Libraries were prepared using the Ovation Ultralow System V2 (Nugen, 0344) and sequenced on the NextSeq 500 (Illumina) with 76 bp single end reads at the John Innes Centre, Norwich, UK.

Library strategy

ChIP-Seq

Library source

genomic

Library selection

ChIP

Instrument model

Illumina NextSeq 500

Data processing

ChIP-seq: Reads were mapped to TAIR10 using Bowtie2-2.3.4.1, retaining mononucleosomal fragments with using parameters -I 130 -X 200. Mapped reads were sorted and indexed using SAMtools-1.9.
ChIP-seq: Bigwig files were generated by normalising IP bam files to respective inputs with the bamCompare tool from deepTools-3.1.1. Bigwig files of enrichment were generated at single base resolution (w1) and at 50 bp intervals (w50).
ChIP-seq: To generate peaks, H2B.8 enrichment was calculated over 50 bp windows and those with > 0.5 log2(IP/input) were retained. Windows within 501 bp were merged using BEDtools-2.28.0. Regions were filtered by size, with those < 201 bp removed from analysis. H2B.8 enrichment was then calculated over the new regions, those with < 0.5 log2(IP/input) were discarded. The remaining regions were defined as H2B.8 peaks.
ChIP-seq: Overlaps with genes and TEs were determined using BEDtools-2.28.0; 50% of the feature was required to be overlapped by a peak to be defined as an overlap.
RNA-seq: Adapters were trimmed using TrimGalore-0.4.2.
RNA-seq: For differential expression analysis, reads were mapped to TAIR10 with TopHat-2.0.10. Kallisto-0.43.0 was used to obtain TPM (Transcripts Per Million) values. Pairwise statistical analysis was undertaken with Sleuth-0.30.0 in R-3.6.0. Differentially expressed genes and TEs were identified by log2 fold change of ≥ 2 and q < 0.05.
Hi-C: For Hi-C data assay, clean reads were mapped to TAIR10 genome using the HiC-Pro (version 2.11.1) pipeline. The bam files (bwt2merged.bam) generated by HiC-Pro containing mapping information were used as input files for Fan-C (version 0.9.8). The module ‘fanc auto’ was applied to generate 500 kb, 100 kb, 50 kb, 10 kb, 1kb contact matrix (hic files).
Assembly: TAIR10
Supplementary files format and content: ChIP-seq: Bigwig files generated with bamCompare in deepTools-3.1.1. Scores at w1 or w50 resolution represent log2(IP/input).
Supplementary files format and content: ChIP-seq: BED file of H2B.8 peaks in sperm and seedling.
Supplementary files format and content: ChIP-seq: BED file of H2B.8 peaks in sperm and seedling.
Supplementary files format and content: ChIP-seq: BED file of genes or TEs marked by H2B.8 in sperm and seedling.
Supplementary files format and content: RNA-seq: Matrix (.txt) file of WT and h2b.8 sperm cell replicate TPM values, fold change and q values for genes or TEs.
Supplementary files format and content: RNA-seq: Matrix (.txt) file of WT and p35S::H2B.8-eGFP seedling replicate TPM values, fold change and q values for genes or TEs.
Supplementary files format and content: RNA-seq: Matrix (.txt) file of drought treated and respective control WT and p35S::H2B.8-eGFP seedling replicate TPM values, fold change and q values for genes.
Supplementary files format and content: Hi-C: matrix and regions files of different resolution generated by Fan-C contain genomic contact and corresponding index information, respectively.

Submission date

Mar 09, 2022

Last update date

Jul 26, 2022

Contact name

Xiaoqi Feng

E-mail(s)

xiaoqi.feng@jic.ac.uk

Organization name

John Innes Centre

Department

Cell and Developmental Biology