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| Status |
Public on Sep 05, 2020 |
| Title |
CTRL004 S3 fraction |
| Sample type |
SRA |
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| Source name |
normal control fibroblasts
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| Organism |
Homo sapiens |
| Characteristics |
cell type: patient derived skin fibroblast
passage: 20
fraction: S3
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| Growth protocol |
Primary fibroblast cell lines were cultured in DMEM High glucose with glutamax supplemented with 15% FBS and 1% Pen/Strep.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
4 million fibroblasts were washed in PBS 1X, and extracted in cytoskeleton buffer (CSK: 10 mM PIPES pH 6,8; 100 mM NaCl; 1 mM EGTA; 300 mM Sucrose; 3 mM MgCl2; 1X protease Inhibitors by Roche Diagnostics; 1 mM PMSF) supplemented with 1 mM DTT and 0,5% Triton X-100. After 5 min at 4°C the cytoskeletal structure was separated from soluble proteins by centrifugation at 3000 rpm for 3 min, and the supernatant was labeled as S1 fraction. The pellets were washed with an additional volume of cytoskeleton buffer. Chromatin was solubilized by DNA digestion with 25U of RNase–free DNase (Invitrogen) in CSK buffer for 30 min at 37°C. To stop digestion, ammonium sulphate was added in CSK buffer to a final concentration of 250 mM and, after 5 min at 4°C samples were pelleted at 5000 rpm for 3 min at 4°C and the supernatant was labeled as S2 fraction. After a wash in CSK buffer, the pellet was further extracted with 2M NaCl in CSK buffer for 5 min at 4°C, centrifuged at 5000 rpm 3 min at 4°C and the supernatant was labeled as S3 fraction. This treatment removed the majority of histones from chromatin. After 2 washing in NaCl 2M CSK, the pellets were solubilized in 8M urea buffer to remove any remaining protein component by applying highly denaturing conditions. This remaining fraction was labeled as S4.
For DNA fractionation, we took an aliquot corresponding to 30% of volume of S2, S3, S4 fractions and added TE buffer to reach a final volume of 300μL. After incubation with RNAse A (Roche) (90 minutes at 37°) and Proteinase K (Sigma) (150 minutes at 55°), DNA was extracted from beads by standard phenol/chloroform extraction, precipitated and resuspended in 25μl milliQ H2O. After Nanodrop (260/280 = 1,7-1,9; 260/230 ≥ 2) and Qubit HS DNA quantification, we added H2O to a final volume of 105μL. Then samples were transferred to 96 well plates and sonicated 4 times with Bioruptor sonicator (10 minutes 30 seconds ON - 30 seconds OFF, High Power). The DNA profiles were finally checked by capillary electrophoresis (Agilent 2100 Bioanalyzer). Finally, DNA libraries were prepared by using NuGEN Ovation Ultralow Library Prep System kit and then sequenced using an Illumina HiSeq 2500 instrument according to manufacturer’s instructions (Illumina).
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 2500 |
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| Data processing |
library strategy: Sequential Analysis of MacroMolecules accessibilitY (SAMMY-seq)
Reads were aligned to the hg38-noalt reference human genome available in the bcbio-nextgen pipeline, using bwa aln (version 0.7.12) with options -n 2 -k 2 and saved the results in sam format with bwa samse.
The sam files were converted to bam and name sorted with samtools (version 1.3.1). We marked PCR duplicates using the biobambam2 toolset (version 2.0.54). We discarded reads mapping to non-autosomal chromosomes, PCR duplicates, qcfail, multimapping and unmapped reads with samtools.
We calculated the genome wide IP - input signal for all samples, using the SPP package (version 1.15.4). We imported bam files into the R (version 3.3.1) statistical environment, and selected informative reads with the get.binding.characteristics and select.informative.tags functions, removed anomalous positions with extremely high number of reads using the remove.local.tag.anomalies function, and calculated the differential signal, smoothed by a Gaussian kernel, using the get.smoothed.tag.density function with the default bandwidth and tag.shift = 0 parameters.
We used the Enriched Domain Detector (EDD) tool to select significantly enriched SAMMY-seq domains by comparing less accessible fractions to more accessible ones (S3 vs S2, S4 vs S3 and S4 vs S2 comparisons) in each sample, with the following options: --gap-penalty 25 --bin-size 50 --write-log-ratios --write-bin-scores and also excluding blacklisted genomic regions containing telomeric, centromeric, and certain heterochromatic regions62. We also changed the required_fraction_of_informative_bins parameter to 0.98.
Genome_build: hg38-noalt
Supplementary_files_format_and_content: bed: SAMMY-seq enriched domain coordinates for a specific sample. bigWig: genome wide IP - input signal for a specific sample, smoothed by a Gaussian kernel.
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| Submission date |
Aug 16, 2018 |
| Last update date |
Sep 05, 2020 |
| Contact name |
Chiara Lanzuolo |
| E-mail(s) |
[email protected]
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| Phone |
0200660358
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| Organization name |
CNR and Istituto Nazionale Genetica Molecolare
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| Lab |
Lanzuolo Lab
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| Street address |
Via Francesco Sforza 35
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| City |
Milan |
| State/province |
--- |
| ZIP/Postal code |
20122 |
| Country |
Italy |
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| Platform ID |
GPL16791 |
| Series (1) |
| GSE118633 |
SAMMY-seq, H3K9me3 and H3K27me3 ChIP-seq and RNA-seq of control and progeria fibroblasts |
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| Relations |
| BioSample |
SAMN09727150 |
| SRA |
SRX4475554 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM3335764_CTRL004_S3_vs_S2.bed.gz |
1.3 Kb |
(ftp)(http) |
BED |
| GSM3335764_CTRL004_S3_vs_S2.bigwig |
499.8 Mb |
(ftp)(http) |
BIGWIG |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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