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| Status |
Public on Apr 26, 2021 |
| Title |
INPUT-93T449-BG4_rep2 |
| Sample type |
SRA |
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| Source name |
93T449 (ATCC #CRL-3043)
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| Organism |
Homo sapiens |
| Characteristics |
cell line: 93T449
genotype: amp12q14-15
antibody: none
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| Growth protocol |
93T449 cells were seeded in T75 flasks in the presence of RPMI 1640 Medium supplemented with 10 % FBS and grown to 80 % confluence.
93T449 cells were routinely subcultured with a 1:2 to 1:4 ratio using Trypsin-EDTA solution and typically kept into a T75 flask of uncoated plastic in RPMI 1640 medium (Gibco, Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% heat inactivated FBS.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
2 million cells were fixed in RPMI containing 1 % (v/v) formaldehyde (Thermo Scientific™ Pierce™ #28906) and 10 % (v/v) FBS for 10 min at RT. After 5 min quenching with 125 mM glycine, cells were pelleted and washed twice with PBS containing 10 % FBS. The flash frozen pellets were lysed for 5 min on ice in 100 μl of 50 mM tris-HCl pH 8.0, 10 mM EDTA, 0.5 % SDS and protease inhibitor cocktail. Samples were sonicated using the Covaris E220 to shear chromatin to an average size of 100-500 bp (2 % duty cycle, 105 W peak incident power, 200 cycles per burst, 25 min). Sheared chromatin was diluted 1:5 in IP-buffer (10 mM Tris-HCl pH 7.5, 1mM EDTA, 0.5 mM EGTA, 1% Triton X-100, 0.1 % SDS, 0.1 % Na-deoxycholate and 140 mM NaCl) supplemented with protease inhibitor cocktail. After centrifuging 10 min at 13,000g at 4 °C, the supernatant containing soluble chromatin fraction was recovered and incubated with 0.7 mg/ml RNase A (Thermo Scientific™ #EN0531) for 30 min at 37 °C. For chromatin immunoprecipitation 10 μl protein-G magnetic beads (Thermo Scientific™ Pierce™ #88848) were washed in IP-buffer and incubated with 1 μg Anti-FLAG Ab (Sigma-Aldrich #F3165) for 1 h at 4 °C on a rotating wheel. 50 μl of RNA digested chromatin were incubated with 250 ng BG4 Ab (or without for the mock negative control) for 1h at 16 °C. The anti-FLAG coated beads were washed with IP-buffer and incubated with chromatin-BG4 complex for 3 h at 4 °C on a rotating wheel. Beads were washed 4 times with IP-buffer and once in wash buffer (10 mM Tris-HCl pH 8.0, 10 mM EDTA). Elution of immonoprecipitates and chromatin crosslink reversal were performed incubating beads with 70 μl elution buffer (10 mM Tris-HCl pH 8.0, 5 mM EDTA, 300 mM NaCl and 0.5 % SDS) containing 0.3 mg/ml RNase A (Thermo Scientific™ Pierce™ #EN0531) for 30 min at 37 °C followed by the addition of 0.5 mg/ml proteinase K for 1h at 55 °C and 8 h at 65 °C shaking. Supernatant was then recovered and incubated for 1 additional h at 55 °C in the presence of 0.25 mg/ml proteinase K (Thermo Scientific™ #EO0491). The eluate was finally purified with SPRI AMPure XP beads (Beckman Coulter #A63881).
NEBNext Ultra II DNA library Prep Kit for Illumina, NEB
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 1500 |
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| Description |
93T449 cell line was established from a well-differentiated liposarcoma from the retroperitoneum of a 68 year old female.
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| Data processing |
reads were mapped to the human genome (hg38) using bowtie (Langmead et al., 2009).
Genome_build: hg38
Supplementary_files_format_and_content: bigWig files were generated from Homer tag directories using makeBigWig.pl
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| Submission date |
Feb 19, 2020 |
| Last update date |
Apr 26, 2021 |
| Contact name |
Filippo M. Cernilogar |
| E-mail(s) |
filippo.cernilogar@med.uni-muenchen.de
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| Organization name |
LMU Munich
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| Department |
Biomedical Center
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| Street address |
Grosshaderner Strasse 9
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| City |
Planegg-Martinsried |
| ZIP/Postal code |
82152 |
| Country |
Germany |
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| Platform ID |
GPL18460 |
| Series (2) |
| GSE145535 |
DNA G-quadruplexes modulate highly transcribed genes in human chromatin [ChIPseq] |
| GSE145543 |
DNA G-quadruplexes modulate highly transcribed genes in human chromatin |
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| Relations |
| BioSample |
SAMN14135872 |
| SRA |
SRX7749596 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4320549_ChIPseq_Input_BG4_rep2_93T449_m1.ucsc.bigWig |
209.9 Mb |
(ftp)(http) |
BIGWIG |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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