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| Status |
Public on Feb 15, 2024 |
| Title |
h1_seedling_Hi-C |
| Sample type |
SRA |
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| Source name |
h1 seedling
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| Organism |
Arabidopsis thaliana |
| Characteristics |
ecotype: Col-0
genotype: h1
tissue: ten-day-old seedlings
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| Growth protocol |
Plants were grown under long day (16 hr light; 8 hr dark) conditions at 22 °C.
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| 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.
The Hi-C library construction and sequencing were conducted by Annoroad Gene Technology Co., Ltd (Beijing, China). Briefly, The pelleted nuclei were washed twice with 1x ice cold CutSmart buffer and finally resuspended in 0.5 ml volume. SDS was applied to permeabilize nuclei at 65 °C for 10 min, Triton X-100 was added to quench SDS. Thereafter, chromatin was digested with 400 units MboI overnight at 37 °C with gentle rocking. MboI was then denatured to cease activity. Digested chromatin underwent DNA end repair with biotin-14-dCTP insertion followed by blunt-end ligation. After decrosslinking with proteinase K at 65 °C, DNA was purified by phenol chloroform extraction method. Biotin-14-dCTP was removed from non-ligated DNA fragment ends using T4 DNA polymerase. DNA was sheared to a range of 200 to 600 bp by sonication. Next, the fragments underwent end repair and were pulled down by streptavidin C1 magnetic beads to enrich for fragments containing contact information. Fragment ends were then A-tailed, sequencing adapters were ligated, and libraries were amplified by PCR for 12-14 cycles. Following purification, libraries were sequenced using the Illumina HiSeq platform with 2×150 bp length.
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| Library strategy |
Hi-C |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
HiSeq X Ten |
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| Description |
h1_HiC
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| Data processing |
MNase-seq: Reads were mapped to TAIR10 using Bowtie2-2.3.4.1, retaining mononucleosomal fragments with using parameters -I 130 -X 200. NuMap was then performed to compute nucleosome positions.
Bisulfite-Seq: We used Perl scripts to convert all the Cs in the reads (and in the scaffolds) to Ts, and aligned the converted reads to the converted reference scaffold, allowing up to two mismatches per read.
w1: We used Perl scripts to recover the original sequence of each mapped read and, for each C (on either strand), count the number of times it was sequenced as a C or a T.
w50: We used a Perl script to calculate fractional methylation (#C/(#C+#T)) within a 50 bp sliding window for each sequence context (CG, CHG, CHH).
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 and 25 kb contact matrix (hic files).
Genome_build: TAIR10
Supplementary_files_format_and_content: MNase-seq: BED files of nucleosome positions.
Supplementary_files_format_and_content: Bisulfite-Seq: GFF files of fractional methylation data either for individual cytosines (w1) or in 50 bp windows (w50).
Supplementary_files_format_and_content: Hi-C: matrix and regions files of different resolutions generated by Fan-C contain genomic contacts and corresponding index information, respectively.
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| Submission date |
Jun 10, 2021 |
| Last update date |
Feb 15, 2024 |
| Contact name |
Xiaoqi Feng |
| E-mail(s) |
xiaoqi.feng@jic.ac.uk
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| Organization name |
John Innes Centre
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| Department |
Cell and Developmental Biology
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| Lab |
Xiaoqi Feng
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| Street address |
Norwich Research Park
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| City |
Norwich |
| State/province |
Norfolk |
| ZIP/Postal code |
NR4 7UH |
| Country |
United Kingdom |
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| Platform ID |
GPL23157 |
| Series (1) |
| GSE176526 |
Linker histone H1 drives heterochromatin condensation via phase separation in Arabidopsis |
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| Relations |
| BioSample |
SAMN19655044 |
| SRA |
SRX11110862 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM5368665_h1_seedling_25kb.matrix.txt.gz |
110.8 Mb |
(ftp)(http) |
TXT |
| GSM5368665_h1_seedling_25kb.regions.txt.gz |
17.8 Kb |
(ftp)(http) |
TXT |
| GSM5368665_h1_seedling_500kb.matrix.txt.gz |
328.9 Kb |
(ftp)(http) |
TXT |
| GSM5368665_h1_seedling_500kb.regions.txt.gz |
837 b |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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