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| Status |
Public on May 13, 2024 |
| Title |
2M-rep1, C2C12, S2S |
| Sample type |
SRA |
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| Source name |
C2C12
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| Organism |
Mus musculus |
| Characteristics |
cell line: C2C12
cell type: Myoblasts
number of_cells: 2M
fraction: S2S
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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% CO2 in DMEM high glucose supplemented with 10% FBS
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
For 4f-SAMMY-seq, 2 million C2C12 myoblasts were detached from the culture plate by 3min incubation in trypsin-EDTA at 37°C, 5% CO2. After two washes in cold PBS, they were resuspended in 600μL of CSK-Triton buffer (10mM PIPES pH 6.8, 100mM NaCl, 1mM EGTA, 300mM Sucrose, 3mM MgCl2, 1mM PMSF, 1 mM DTT, 0.5% Triton X-100, with protease inhibitors). After 10min incubation on a wheel at 4°C, soluble proteins and the cytoskeletal structure were separated from the nuclei by centrifugation at 900g for 3min 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 100μL of CSK buffer (10mM PIPES pH 6.8, 100mM NaCl, 1mM EGTA, 300mM Sucrose, 3mM MgCl2, 1mM PMSF, with protease inhibitors) and incubated for 60min at 37°C with 25U of RNase–free DNase I (Invitrogen, AM2222). To stop DNA digestion, ammonium sulphate was added in the CSK buffer to a final concentration of 250mM. After 5min incubation on ice, the sample was pelleted at 900g for 3min at 4°C; the supernatant, containing digested chromatin fragments, was labeled as S2 fraction. Afterwards, the pellet was washed with 200μL of CSK buffer, resuspended in 100μL of CSK-NaCl buffer (CSK buffer with NaCl final concentration increased to 2M) and then incubated 10min on a wheel at 4°C. At the end of the incubation, the sample was centrifuged at 2300g for 3min at 4°C and the supernatant was labeled as S3 fraction. Finally, after two washes in 200μL of CSK-NaCl buffer, the pellet was solubilized in 100μL of 8M urea; the final suspension was labeled as S4 fraction. Half volume of S2 and the the whole volume of S3 and S4 were diluted in TE (10mM TrisHCl pH 7.5, 1mM EDTA pH 8.0) to a final volume of 200μl and then incubated 90min at 37°C with 6μL of RNAse cocktail (Ambion, AM2286), followed by 150min 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) extraction, precipitated in 70% ethanol, 0.3 M sodium acetate and 20 μg glycogen overnight at -20°C or 1 hour in dry ice and resuspended in nuclease-free water. S2 was additionally purified using PCR DNA Purification Kit (Qiagen, 28106) and then DNA fragments in this fraction were separated using AMPure XP paramagnetic beads (Beckman Coulter, A63880) to obtain S2S (<300bp) and S2L (> 300bp) fractions. In detail, beads were added to the S2 fraction in a 0.95x (v/v) ratio, so to bind fragments larger than 300bp while leaving smaller fragments in solution. 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 the 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 resuspending in water, they were also detached from the beads (S2S fraction). After DNA isolation, S2L, S3 and S4 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 200bp (settings: water bath 20°C, peak power 30.0, duty factor 20.0, cycles/burst 50; duration: 125sec for S2 and S2L, 175sec for S3 and S4). DNA in the fractions was then quantified using Qubit dsDNA HS Assay Kit (Invitrogen, Q32854) and a Qubit 4.0 fluorometer; quality control was performed by run on an Agilent 2100 Bioanalyzer System using the High Sensitivity DNA Kit (Agilent, 5067-4626).
Libraries were 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 |
Illumina HiSeq 2000 |
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| Description |
4f
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| Data processing |
Trimming. High-throughput sequencing 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).
Alignment. After trimming, 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 mm9 genomeand 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). In 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.
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 2308125349 bp and we excluded regions known to be problematics in term of sequencing reads coverage using the black list f rom the following work : https://doi.org/10.1038/s41598-019-45839-z
Assembly: mm9
Supplementary files format and content: Bigwiggle files (.bw) are created using Deeptools for genomic track data
Library strategy: SAMMY-seq
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| Submission date |
Mar 21, 2024 |
| 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 |
GPL13112 |
| Series (2) |
| GSE262103 |
Biochemical properties of chromatin domains define genome compartmentalization [III] |
| GSE262104 |
Biochemical properties of chromatin domains define genome compartmentalization |
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| Relations |
| BioSample |
SAMN40563428 |
| SRA |
SRX24010599 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM8157581_2M-rep1_C2C12_S2S.bw |
124.4 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
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