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Sample GSM5941849 Query DataSets for GSM5941849
Status Public on Jul 26, 2022
Title H2B8_seedling_Hi-C_rep2
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
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 were harvested and fixed with 20 ml 2% formaldehyde solution for 15 min in vacuum conditions at room temperature and then quenched by adding 2.162 ml 2.5 M glycine. Fixed seedling tissue was rinsed with water three times and dried with tissue paper. The nuclei were released by grinding in liquid nitrogen and then resuspended with 25 ml of extraction buffer I (0.4 M sucrose, 10 mM Tris HCl pH 8, 10 mM MgCl2, 5 mM β-mercaptoethanol, 0.1 mM PMSF, 13 μl protease inhibitor). Nuclei were filtered through miracloth (Calbiochem) and then centrifuged at 4000 rpm for 20 min at 4 °C. The supernatant was discarded whilst the pellet was resuspended with 1 ml of extraction buffer II (0.25 M sucrose, 10 mM Tris HCl pH 8, 10 mM MgCl2, 1% Triton X-100, 5mM β-mercaptoethanol, 0.1mM PMSF, 13 μl protease inhibitor). Then, the mixture was centrifuged at 14000 rpm for 10 min at 4 °C and the pellet was resuspended with 300 μl of extraction buffer III (1.7 M sucrose, 10 mM Tris HCl pH 8, 0.15% Triton X-100, 2 mM MgCl2, 5 mM β-mercaptoethanol, 0.1 mM PMSF, 1 μl protease inhibitor), load the mixture onto the top of an equal amount of clean extraction buffer III, then centrifuge at 14000 rpm for 10 min.
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 Hi-C
Library source genomic
Library selection other
Instrument model HiSeq X Ten
 
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
Lab Xiaoqi Feng
Street address Norwich Research Park
City Norwich
State/province Norfolk
ZIP/Postal code NR4 7UH
Country United Kingdom
 
Platform ID GPL23157
Series (1)
GSE161366 Histone H2B.8 compacts flowering plant sperm through chromatin phase separation
Relations
BioSample SAMN26543081
SRA SRX14434541

Supplementary file Size Download File type/resource
GSM5941849_H2B8_seedling_rep2_100kb.matrix.txt.gz 7.9 Mb (ftp)(http) TXT
GSM5941849_H2B8_seedling_rep2_100kb.regions.txt.gz 4.0 Kb (ftp)(http) TXT
GSM5941849_H2B8_seedling_rep2_10kb.matrix.txt.gz 264.1 Mb (ftp)(http) TXT
GSM5941849_H2B8_seedling_rep2_10kb.regions.txt.gz 57.5 Kb (ftp)(http) TXT
GSM5941849_H2B8_seedling_rep2_1kb.matrix.txt.gz 786.9 Mb (ftp)(http) TXT
GSM5941849_H2B8_seedling_rep2_1kb.regions.txt.gz 578.3 Kb (ftp)(http) TXT
GSM5941849_H2B8_seedling_rep2_500kb.matrix.txt.gz 328.6 Kb (ftp)(http) TXT
GSM5941849_H2B8_seedling_rep2_500kb.regions.txt.gz 854 b (ftp)(http) TXT
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file
Processed data are available on Series record

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