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Sample GSM4320549 Query DataSets for GSM4320549
Status Public on Apr 26, 2021
Title INPUT-93T449-BG4_rep2
Sample type SRA
Source name 93T449 (ATCC #CRL-3043)
Organism Homo sapiens
Characteristics cell line: 93T449
genotype: amp12q14-15
antibody: none
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.
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
Library strategy ChIP-Seq
Library source genomic
Library selection ChIP
Instrument model Illumina HiSeq 1500
Description 93T449 cell line was established from a well-differentiated liposarcoma from the retroperitoneum of a 68 year old female.
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
Submission date Feb 19, 2020
Last update date Apr 26, 2021
Contact name Filippo M. Cernilogar
Organization name LMU Munich
Department Biomedical Center
Street address Grosshaderner Strasse 9
City Planegg-Martinsried
ZIP/Postal code 82152
Country Germany
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
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 SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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