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| Status |
Public on May 13, 2024 |
| Title |
C004-r2_4f_S3 (single-end) |
| Sample type |
SRA |
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| Source name |
skin fibroblast
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| Organism |
Homo sapiens |
| Characteristics |
cell type: patient derived skin fibroblast
passage: 19
fraction: S3
protocol: 4f
sample: C004
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| Treatment protocol |
No treatment has been performed on these cells.
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| Growth protocol |
Cells were cultured at 37 °C, 5% CO2in DMEM high glucose supplemented with GlutaMAX(Gibco,10566-016), with further addition of 15% (v/v) FBS (Gibco, 10270106), 100 U/mL penicillin G and 100 μg/mLStreptomycin Sulfate.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
For 4f-SAMMY-seq, 3 million fibroblasts were detached from the culture plate by 3 min incubation in trypsin-EDTA at 37°C, 5% CO2. After two washes in cold PBS, they were resuspended in 600 μL ofCSK-Triton buffer (10 mM PIPES pH 6.8, 100 mM NaCl, 1 mM EGTA, 300 mM Sucrose, 3 mM MgCl2, 1mM PMSF, 1 mM DTT, 0.5% Triton X-100, with protease inhibitors). After 10 min incubation on a wheel at 4°C, soluble proteins and the cytoskeletal structure were separated from the nuclei by centrifugation at 900g for 3 min at 4°C; the supernatant was labeled as S1 fraction. The pellet was then washed with an additional volume of CSK-Triton buffer, resuspended in 100 μL of CSK buffer (10 mM PIPES pH 6.8, 100 mMNaCl, 1 mM EGTA, 300 mM Sucrose, 3 mM MgCl2, 1mM PMSF, with protease inhibitors) and incubated for 60 min at 37°C with 25 U of RNase–free DNase I (Invitrogen, AM2222). To stop DNA digestion, ammonium sulphate was added in the CSK buffer to a final concentration of 250 mM. After 5 min incubation on ice, the sample was pelleted at 900g for 3 min at 4°C; the supernatant, containing digested chromatin fragments, waslabeled as S2 fraction. Afterwards, the pellet was washed with 200 μL of CSK buffer, resuspended in 100 μL of CSK-NaCl buffer (CSK buffer with NaCl final concentration increased to 2 M) and then incubated 10 min on a wheel at 4°C. At the end of the incubation, the sample was centrifuged at 2300g for 3 min at 4°C and the supernatant was labeled as S3 fraction. Finally, after two washes in 200 μL of CSK-NaCl buffer, the pellet was solubilized in 100 μL of 8M urea; the final suspension was labeled as S4 fraction. For 10kh-SAMMY-seq, 10 thousand fibroblasts were treated following 4f-SAMMY-seq protocol, halving the volume in each step and using 12.5U of DNAse I (Invitrogen, AM2222); in this way we achieved a 150 fold increase in the ratio of enzyme units per starting number of cells, while maintaining the DNase concentration unaltered. Fractions S2, S3 and S4 were diluted in TE (10 mM TrisHCl pH 7.5, 1mM EDTA pH 8.0) to a final volume of 200 μl and then incubated 90 min at 37°C with 6 μL of RNAse cocktail (Ambion, AM2286), followed by 150 min at 55° with Proteinase K (Invitrogen, AM 2548) to a final concentration of 0.2 μg/μL. Next, DNA was isolated through phenol:chloroform:isoamyl alcohol (Sigma, 77617), precipitated and resuspended in nuclease-free water. On the next day, S2 from 4f-SAMMY-seq was additionally purified using PCR DNA Purification Kit (Qiagen, 28106) and DNA fragments in this fraction were separated using AMPure XP paramagnetic beads (Beckman Coulter, A63880) to obtain S2S (< 300 bp) and S2L (> 300 bp) fractions. Beads were added to the S2 fraction in a 0.95x (v/v) ratio, so to bind fragments larger than 300bp. Magnetic separation of beads from supernatant allowed the physical separation of larger fragments (on the beads) from shorter ones (in the supernatant). Larger fragments bound on beads were then washed in 85% ethanol, resuspended in water and magnetically separated from the beads (S2L fraction). Shorter fragments in the supernatant of the first step were bound to beads by adding a further 0.85x (v/v) beads ratio to the suspension; after washing in 85% ethanol and resuspension in water, they were also detached from beads (S2S fraction). Afterwards, S2 (from 10kh-SAMMY-seq), S2L (from 4f-SAMMY-seq), S3 and S4 (from both 10kh- and 4f-SAMMY-seq) fractions were transferred to screw cap microTUBEs (Covaris, 004078) and sonicated in a Covaris M220 focused-ultrasonicator to obtain a smear of DNA fragments peaking at 200 bp (settings: water bath 20°C, peak power 30.0, duty factor 20.0, cycles/burst 50; duration: 125 sec for S2 and S2L, 175 sec for S3 and S4). Fractions were quantified using Qubit dsDNA HS Assay Kit (Invitrogen, Q32854 ) and a Qubit 4.0 fluorometer; quality control was performed by running them on an Agilent 2100 Bioanalyzer System using the High Sensitivity DNA Kit (Agilent, 5067 – 4626).
Libraries were then created from each fraction using the NEBNext Ultra II DNA Library Prep Kit for Illumina (NEB, E7645L) and the Unique Dual Index NEBNext Multiplex Oligos for Illumina (NEB, E6440S).
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
NextSeq 2000 |
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| Data processing |
Trimming. High - throughput s equencing reads were trimmed using Trimmomatic (v0.39) (Bolger, Lohse, and Usadel 2014) using the following parameters: 2 for seed_mismatch, 30 for palindrome_thresh old, 10 for simple_threshold, 3 for leading, 3 for trailing and 4:15 for sliding window. The sequence minimum length threshold of 35 was applied to all data sets . We used the Trimmomatic “TruSeq3 - SE.fa” (for single end) and “TruSeq3 - PE - 2.fa” (for paired end) as clip files .
Alignment. After trimm ing, reads were aligned using BWA (v0.7.17 - r1188) (Li and Durbin 2009) setting – k parameter as 2 and using as reference genome the UCSC hg38 genome (only canonical chromosomes were used ) and the output saved in BAM file format .
Filtering. The PCR duplicates were marked with Picard (v2.22; https://github.com/broadinstitute/picard) MarkDupuplicates option , then filtered using Samtools (v1.9) (Li et al. 2009) . I n addition, we filtered all the reads with mapping quality lower than 1. Each sequencing lane was analyzed separately and then merged at the end of the process. For C002 - rep1 and C004 - rep2 two sequencing runs from the same library were produced and merged after alignment and filtering.
Genomic tracks. To compute reads distribution profile (genomic tracks) we used Deeptools (v3.4.3) (Ramirez et al. 2016) bam Coverage function. For these analyses the genome was binned at 50bp, the reads extended up to 250 bp and RPKM normalization method was used. We considered a genome size of 2701495761 bp (value suggested in the Deeptools manual https://deeptools.readthedocs .io/en/latest/content/feature/effectiveGenomeSize.html) and we excluded regions known to be problematics in term of sequencing reads coverage using the black list f rom the ENCODE portal ( https://www.encodeproject.org/files/ENCFF356LFX/ )
Comparisons. for relative comparisons (relative enrichment , i.e. log2 normalized ratio) between SAMMY - seq fraction s we us ed the SPP R package (v1.16.0) (Kharchenk o, Tolstorukov, and Park 2008) and R statistical environment (v3.5.2). The reads were imported from the BAM files using the “read.bam.tags” function, then filtered using “remove.local.tag.anomalies” and finally the comparison s performed using the function “get.smoothed.enrichment.mle” setting “tag.shift = 0” and “background.density.scaling = TRUE”. The resulting enrichment signal correspond s to a log2 normalized ratio between the pair of sequencing samples.
Assembly: hg38
Supplementary files format and content: Bigwiggle files (.bw) are created using Deeptools for genomic track data and with wigToBigWig (v4) starting from wiggle files generated by SPP for what concern comparisons.
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| Submission date |
Mar 06, 2023 |
| Last update date |
May 13, 2024 |
| Contact name |
Chiara Lanzuolo |
| E-mail(s) |
lanzuolo@ingm.org
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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 |
GPL30173 |
| Series (2) |
| GSE226718 |
Biochemical properties of chromatin domains define their compartmentalization [I] |
| GSE262104 |
Biochemical properties of chromatin domains define genome compartmentalization |
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| Relations |
| BioSample |
SAMN33602825 |
| SRA |
SRX19577996 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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